ЕКСПРЕС ДІАГНОСТИКА ФЛАВОБАКТЕРІОЗУ РИБ МЕТОДОМ ПОЛІМЕРАЗНОЇ ЛАНЦЮГОВОЇ РЕАКЦІЇ
The method of rapid identification of fish flavobacteriosis using polymerase chain reaction (PCR) was developed. Specifity and efficiency of the selected oligonucleotide primers was confirmed. Nine Flavobacterium strains were isolated from clinically healthy and infected with Flavobacteriosis rainbo...
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Institute of Agrocultural Microbiology and Agro-industrial Manufacture of NAAS of Ukraine
2014
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| author | Рудь, Ю. П. |
| author_facet | Рудь, Ю. П. |
| author_institution_txt_mv | [
{
"author": "Ю. П. Рудь",
"institution": "Інститут рибного господарства НААН"
}
] |
| author_sort | Рудь, Ю. П. |
| baseUrl_str | https://smic.in.ua/index.php/journal/oai |
| collection | OJS |
| datestamp_date | 2026-07-22T10:10:44Z |
| description | The method of rapid identification of fish flavobacteriosis using polymerase chain reaction (PCR) was developed. Specifity and efficiency of the selected oligonucleotide primers was confirmed. Nine Flavobacterium strains were isolated from clinically healthy and infected with Flavobacteriosis rainbow trout Onchorhynchus mykiss. Flavobacterium columnare strain was identified using the developed rapid diagnosis method. Phylogenetic analysis had indicated the kinship of isolated strains to Flavobacterium genera. Neither F. psychrophylum or F. branchiophilum strains were identified in the isolated samples. The species identification capacity using the 16S rRNA gene restriction analysis was shown. The developed PCR method can be used for rapid identification of different fish flavobacteriosis forms in fish farms in Ukraine. |
| doi_str_mv | 10.35868/1997-3004.18.132-145 |
| first_indexed | 2025-07-17T12:24:22Z |
| format | Article |
| fulltext |
RAPID DIAGNOSTIC OF FISH FLAVOBACTERIOSIS USING POLYMERASE
CHAIN REACTION
Rud Yu. P.
Institute of Fisheries, NAAS of Ukraine
135, Obukhivska str., Kyiv, 03164, Ukraine
e-mail: rud_yuriy@ifr.com.ua
The method of rapid identification of fish flavobacteriosis using polymerase chain
reaction (PCR) was developed. Specifity and efficiency of the selected oligonucleotide
primers was confirmed. Nine Flavobacterium strains were isolated from clinically
healthy and infected with Flavobacteriosis rainbow trout Onchorhynchus mykiss.
Flavobacterium columnare strain was identified using the developed rapid diagnosis
method. Phylogenetic analysis had indicated the kinship of isolated strains to
Flavobacterium genera. Neither F. psychrophylum or F. branchiophilum strains were
identified in the isolated samples. The species identification capacity using the 16S rRNA
gene restriction analysis was shown. The developed PCR method can be used for rapid
identification of different fish flavobacteriosis forms in fish farms in Ukraine.
Key words: Flavobacteriosis, rainbow trout, PCR.
The genus Flavobacterium is characterized with gram-negative, aerobic, oxidase-
positive, nonenzymatic, yellow-pigmented and mostly gliding bacteria that do not form
spores [1]. Over the past 10 years Flavobacterium genus was replenished with many new
species and now includes 75 species. These microorganisms inherent such ecological
niches as fresh and brackish waters and soils [2]. Most of Flavobacterium genus
representatives are pathogens or opportunistic infections agents that infect large range of
organisms, including plants, fish and humans [3-5]. F. psychrophylum and F. columnare
are the most hostile representatives of the Flavobacterium genus in fish culture, leading
to the mass death of fish in fish farms [6, 7].
Bacteria F. psychrophylum is the agent of infectious disease of salmon called
“Bacterial cold water disease” (BCWD) [8] and cause liver inflammation in juvenile
rainbow trout fish (Rainbow trout fry disease, RTFS) [9]. These diseases are prevalent in
Scandinavian countries, but can be also found in cold water farms in central and eastern
Europe [10]. The main sign of F. psychrophylum infection is the formation of deep
ulcers on the body surface of infected fish.
Columnaris or cotton-wool disease, caused by bacterial pathogen F. columnare –
is infectious and highly contagious disease, that cause massive loss of freshwater fish
species. This disease develops at a temperature of 15 °C and above and is found in
salmon, channel catfish, carp and others [11]. Often columnaris disease comes out as a
result of secondary or opportunistic infection [12]. The appearance on the fish flesh of
pale whitish areas that resemble balls of wool or cotton are among the disease signs.
During the course of the columnaris disease affected fish gills become of pale shade with
necrotic areas. In juveniles the disease can occur without typical symptoms. Previously it
was thought that this disease is caused by a complex bacterial infection formed by
Flexibacter columnaris, Bacillus columnaris, Chondrococcus columnaris and Cytophaga
columnaris. Also it was mistakenly believed that given disease has fungal etiology [13].
Another flavobacteriosis agent is bacteria F. branchiophilum, causing bacterial gill
disease (BGD) of trout [14]. Besides the above mentioned species, flavobacteriosis and
fish mortality is also associated with other representatives of the Flavobacterium genus,
like, F. johnsoniae, F. aquatile, F. hydatis and F. succinicans [2, 15, 16].
One of the key factors of the rapid development of global aquaculture is the
control of infectious diseases in breeding facilities. In Ukraine the situation with the
distribution and origin of flavobacteriosis in natural waters and fishery enterprises does
not fully investigated. Moreover, this bacterial disease is stated to be one of the most
dangerous in modern aquaculture. The purpose of given research was to isolate bacteria
of Flavobacterium genus from infected and clinically healthy rainbow trout
Onchorhynchus mykiss and develop a rapid diagnostic method of fish flavobacteriosis
using polymerase chain reaction (PCR).
Materials and methods. Initial isolation of microorganisms from juvenile rainbow
trout O. mykiss suspected of flavobacteriosis disease was performed on meat peptonic
agar (MPA). Samples for bacteriological studies were taken from gills, skin and
abdominal cavity. Similar studies were carried out also for clinically healthy rainbow
rainbow trout. Isolation of pure culture, colonies and cell morphology research were
performed by conventional methods [17].
DNA was extracted from the pure cultures colonies. For this, the bacterial
suspensions were prepared. Bacteria were inserted into the 100 µl of phosphate buffer
(137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) with sterile
needle. 500 microliter of lysis buffer (10 mM TRIS-HCl pH 8.0, 0.1 M NaCl, 25 mM
EDTA, 0.5% sodium dodecyl sulfate) and 3 ml of proteinase K (~600 units / µl) were
added, thoroughly mixed and incubated for 1 hour at 37 °C. DNA was extracted with
phenol (pH 8.0) and centrifuged for 5 minutes at 13 400 rpm. The supernatant was
collected and was used for subsequent DNA extraction with a mixture of chloroform -
isoamyl alcohol (24:1). The suspension was centrifuged at microcentrifuge for five
minutes at 13 400 rpm. 0.1 volume of 3 M sodium acetate (pH 5.2) and 2.5 volume of
cooled to -20 °C ethanol were added to supernatant. DNA precipitation was carried out
at room temperature for 1 hour. Then samples were centrifuged at 13 400 rpm for 10
minutes. DNA precipitate was washed with 70% ethanol. Received DNA samples were
than dissolved in deionized water free of nucleases [18].
In order to develop oligonucleotide primers specific to bacteria – agents of
flavobacteriosis, determine their specificity and physical properties the Vector NTI 10
software was used. In addition, primers specificity was tested using the online BLAST
service (www.ncbi.nim.nih.gov/blast). The sequence of primers for F. columnare was
FCF 5'-AAGGCAACGATGGGTAG-3' and FCR 5'-GCACGGAGTTAGCCGATC-3'.
Half-nested PCR was used for F. psychrophylum diagnosis. The following set of
primers: FP1 5'-CTTAGTTGGCATCAACAC-3' and FP3 5'-
ACACTGGCAGTCTTGCTA-3' was used during the first stage of half-nested PCR,
while PSY1 5'- GTTGGCATCAACACACT-3 'and FP3 – during the second stage.
For identification of the Flavobacterium genus bacteria the restriction fragment
length polymorphism method of PCR products (PCR-RFLP) was used [19]. The
amplification of 16S rRNA gene was perfomed with the following primers: 27F 5'-
AGAGTTTGATCMTGGCTCAG-3' and 1492R 5'-CCGYTACCTTGTTACGACTT-3'.
Amplification was performed on amplifier "96 Universal Gradient PEQ STAR»
(PEQLAB, Germany). The reaction mixture had consisted of the following components:
12.5 ml DreamTaqTM Green PCR Master Mix (2X) (Thermo Scientific), oligonucleotide
primers (Metabion, Germany) – 1 mm of each (20 pmol/ml), 1 ml of sample DNA and
sterile deionized water used to bring the total volume to 25 ml. Amplification included 1
cycle of prior denaturation at 94 °C (3 minutes) and 35 cycles of denaturation at 94 °C
(30 seconds), primer annealing at 64 °C (30 seconds), synthesis at 72 °C (1 minute) and
extra synthesis cycle at 72 °C (7 minutes). After PCR the products were analyzed on 2%
agarose gels in TAE buffer (40 mM TRIS-HCl, 20 mM acetic acid, 1 mM EDTA) and
observed in UV transilluminator.
During the restriction analysis of 16S rRNA gene of flavobacteria the restriction
endonucleases HindIII and BamHI (Fermentas) were used. 10 ml amplified DNA, 2 ml
buffer and 1 ml of corresponding restrictions enzyme were inserted into microtube. The
total volume was adjusted to 20 ml with sterile deionized water. The mixture was left to
react overnight in an incubator at 37 °C. Restriction products were analyzed on 2%
agarose gel with the addition of ethidium bromide at a concentration 0.5 g / cm3.
Electrophoresis was performed in TAE buffer (40 mM TRIS-HCl, 20 mM acetic acid, 1
mM EDTA) on the device for horizontal electrophoresis «Scie-Plus» in 100 V mode for
two hours using 100 base pairs of DNA marker (Fermentas). The results were recorded
by ultraviolet radiation photographing in transilluminator UVT1 «Biokom». Analysis of
electrophoregramme was performed using TotalLab TL120 v2008.01software package.
DNA isolation from the gel was performed using a set of Silica Bead DNA Gel
Extraction Kit (Fermentas) according to the manufacturer's protocol. Amplified
fragments were examined on an automatic DNA sequenator 3130 Genetic Analyser
(Applied Biosystems) using a sequencing kit BigDye® Terminator v3.1 Cycle
Sequencing Kit. The nucleotide sequences analysis was performed using ClustalW
algorithm in the software MEGA 5.2 and BLASTN.
Results and discussion. For microbiological studies 9 strains of Flavobacterium
genus bacteria were isolated from clinically healthy and infected with flavobacteriosis
rainbow trout O. mykiss. At MPA flavobacteria were shaped in a bright yellow colonies
of different morphology. At Gram staining all isolated flavobacteria were gram negative.
The cells were rod-shaped with pointed ends.
The results have confirmed that selected oligonucleotide primers specific for F.
columnarae bacteria, had amplified the DNA fragment of expected size. Amplicon size
was about 200 base pairs (Fig. 1). Oligonucleotides specific to F. columnarae were
selected for amplification of 16S rRNA gene fragment. It was established that four out of
nine flavobacteria isolates had belonged to F. columnarae, as evidenced by specific PCR
amplification (Fig. 1). These samples were selected from infected fish with
flavobacteriosis signs, namely in the form of columnaris disease. Gills of infected fish
used for microbiological studies had shown the necrotic signs. Amplification specificity
was tested using nucleotide analysis of PCR products. Sequence results had indicated
that the amplified fragment corresponded to 16S rRNA gene of F. solumnarae bacteria.
It was impossible to diagnose F. psychrophylum bacteria using half-nested PCR.
Given the fact that the symptoms had pointed at flavobacteriosis infection caused by F.
columnarae, none of the samples, including clinically-healthy fish, was not infected with
F. psychrophylum bacteria. It is also should be noted that F. psychrophylum is often
diagnosed in asymptomatic fish, indicating on the circulation of the pathogen in the fish
body [20].
Fig. 1. Amplification of 16S rRNA gene fragment of F. columnarae bacteria: 1-9 –
samples of flavobacteria isolated from rainbow trout O. mykiss; K – control
(amplification fragment of β-actin gene of rainbow trout);
M – DNA marker (FastRuler Low Range DNA Ladder).
In order to identify species belonging of the flavobacteria isolates the PCR-RFLP
was used. After amplification of 16S rRNA gene fragments (Fig. 2) the PCR products
were used for restriction analysis. Analysis of the nucleotide sequences of 16S rRNA
gene of flavobacteria had indicated the possibility of using two restriction endonucleases
HindIII and BamHI for identification of F. psychrophylum, F. branchiophilum and
F. columnarae bacteria. The 16S rRNA gene sequence of F. psychrophylum does not
have restriction sites for HindIII and BamHI, while 16S rRNA gene of F.
branchiophilum, by contrast, was hydrolyzed by both enzymes. The nucleotide sequence
of 16S rRNA gene of F. columnarae has only BamHI restriction site.
Studies indicate that the nucleotide sequence of full-length 16S rRNA gene
amplicons from isolated F. columnarae were restriction sites for BamHI. Amplicons of
16S rRNA gene of other four strains of isolated flavobacteria were also hydrolyzed with
BamHI restriction enzyme. Location of restriction site and size of the products were
identical for all strains of F. columnarae and Flavobacterium sp. (Table 1). It should be
noted that in most flavobacteria, and other bacteria 16S rRNA gene is very conservative
and has about 99% identity. Its downside is that the other Flavobacterium species,
containing only restriction site BamHI cannot be identified using this method. An
analysis of 16S rRNA gene sequences of the Flavobacterium genus representatives, had
shown that other opportunistic flavobacteria except for F. columnarae belong to this
group – F. johnsoniae, F. aquatile, F. hydatis and F. succinicans. The only amplicon of
16S rRNA gene from studied flavobacteria contained HindIII restriction site (Table 1).
Study of nucleotide sequence of the DNA fragment had showed that RT021FU strain of
Flavobacterium sp. belongs to the Flavobacterium oncorhynchi kind (Fig. 3). According
to the literature data F. oncorhynchi is found in normal flora of rainbow trout O. mykiss
[16].
Fig. 2. Amplification of full-length 16S rRNA gene of flavobacteria isolated from
rainbow trout O. mykiss:
1-9 – samples of flavobacteria isolated from rainbow trout O. mykiss; K1 and K2 -
positive controls (DNA of Aeromonas hydrophila and Yersinia ruckeri bacteria,
respectively); K – control (all reaction components except for DNA); M – DNA marker
(FastRuler Low Range DNA Ladder).
Fig. 3. Phylogenetic analysis of 16S rRNA gene of Flavobacterium sp. strain RT021FU,
isolated from rainbow trout O. mykiss. The tree was constructed using the Neighbor-
joining algorithm of MEGA software package version 5.2.
Despite the importance of flavobacteriosis for modern fishery and numerous
investigation of its agents, only a small fraction of the data on pathogen-host interactions
can be found in the literature. Among still unresolved questions remain the colonization
of the skin and gills with flavobacteria, the formation of necrotic ulcers and reasons of
high fish mortality. Diagnosis methods for this disease progresses rapidly and today the
identification of the pathogen with only a few microbial cells is possible. Another
important question coming next to the diagnosis is determination of virulence of
flavobacteria strains. Nucleotide sequencing analysis of the bacteria genomes of the
Flavobacterium genus can help in addressing this issue. Only at the presence of DNA
sequences of flavobacteria, their genes that are responsible for cell virulence the study of
the disease pathogenesis will become possible. Such studies are being performed, but
their effectiveness will increase with the expansion of cooperation between reference
laboratories [10].
Table 1. Restriction analysis of 16S rRNA gene of flavobacteria isolated from rainbow
trout O. mykiss.
Strain Flavobacteria species BamHI HindIII
1 Fl. columnare +
2 Fl. columnare +
3 Fl. columnare +
4 Fl. columnare +
5 Fl. oncorhynchi +
6 Fl. sp. +
7 Fl. sp. +
8 Fl. sp. +
9 Fl. sp. +
Given the commonly increasing phenomenon of antibiotics resistance, research
against flavobacteriosis should be focused on the control of the disease and prevention
maintenance. Strict compliance with the rules of aquaculture, such as compliance with
sanitary and epidemiological norms, fish stocking density and water quality will be the
first steps to make. Vaccines, chemotherapy and probiotics – are also promising areas of
the disease prevention. Perspective is the cultivation of species resistant to
flavobacteriosis. Information about the flavobacteria pathogenesis will allow
development of new methods against this disease that will be economically reasonable
and safe for the environment and aquaculture facilities.
Thus, nine flavobacteria strains were isolated from clinically healthy and infected
with flavobacteriosis rainbow trout O. mykiss. Using the method of express diagnostics
based on PCR the four strains of F. columnarae were identified. Amplification
specificity was tested using the method of nucleotide sequence analysis of amplified
products and by restriction analysis of 16S rRNA gene fragments. The results obtained
have indicated that the developed PCR method can be used for rapid diagnosis of various
forms of fish flavobacteriosis in fishery enterprises in Ukraine. Also, using the restriction
and sequencing detected analyses the bacterial strain F. oncorhynchi was identified,
which is representative of the normal flora of rainbow trout O. mykiss. Using the specific
primers, other agents of flavobacteriosis such as F. psychrophylum and
F. branchiophilum were not identified. For their diagnosis the geography of natural
water resources and fisheries enterprises should be expanded, which will be the subject
of our further research.
|
| id | oai:ojs2.smic.in.ua:article-190 |
| institution | Agriciltural microbiology |
| keywords_txt_mv | keywords |
| language | English Ukrainian |
| last_indexed | 2026-07-23T01:06:11Z |
| publishDate | 2014 |
| publisher | Institute of Agrocultural Microbiology and Agro-industrial Manufacture of NAAS of Ukraine |
| record_format | ojs |
| resource_txt_mv | smicinua/d9/827d0ac8c419e8614b114791d16f09d9.pdf |
| spelling | oai:ojs2.smic.in.ua:article-1902026-07-22T10:10:44Z RAPID DIAGNOSTIC OF FISH FLAVOBACTERIOSIS USING POLYMERASE CHAIN REACTION ЕКСПРЕС ДІАГНОСТИКА ФЛАВОБАКТЕРІОЗУ РИБ МЕТОДОМ ПОЛІМЕРАЗНОЇ ЛАНЦЮГОВОЇ РЕАКЦІЇ Рудь, Ю. П. Flavobacteriosis, rainbow trout, PCR флавобактеріоз, райдужна форель, ПЛР The method of rapid identification of fish flavobacteriosis using polymerase chain reaction (PCR) was developed. Specifity and efficiency of the selected oligonucleotide primers was confirmed. Nine Flavobacterium strains were isolated from clinically healthy and infected with Flavobacteriosis rainbow trout Onchorhynchus mykiss. Flavobacterium columnare strain was identified using the developed rapid diagnosis method. Phylogenetic analysis had indicated the kinship of isolated strains to Flavobacterium genera. Neither F. psychrophylum or F. branchiophilum strains were identified in the isolated samples. The species identification capacity using the 16S rRNA gene restriction analysis was shown. The developed PCR method can be used for rapid identification of different fish flavobacteriosis forms in fish farms in Ukraine. Розроблено метод ідентифікації флавобактеріозу риб за використання полімеразної ланцюгової реакції (ПЛР). Показано специфічність та ефективність підібраних олігонуклеотидних праймерів. Від клінічно здорової та хворої на флавобактеріоз райдужної форелі виділено дев’ять штамів флавобактерій. За допомогою розробленого методу ідентифіковано штам Flavobacterium columnare та шляхом філогенетичного аналізу встановлено його спорідненість з іншими представниками роду Flavobacterium. Інших збудників флавобактеріозу, таких як F. psychrophylum та F. branchiophilum не виявлено. Показана можливість видової ідентифікації флавобактерій методом рестрикційного аналізу гена 16S рРНК. Розроблений метод ПЛР може бути використаний для експрес-діагностики різних форм флавобактеріозу риб в рибогосподарських підприємствах України. Institute of Agrocultural Microbiology and Agro-industrial Manufacture of NAAS of Ukraine 2014-03-31 Article Article Рецензована Стаття application/pdf application/pdf https://smic.in.ua/index.php/journal/article/view/190 10.35868/1997-3004.18.132-145 Agricultural microbiology; Vol. 18 (2014): Agriciltural microbiology; 132-145 Сільськогосподарська мікробіологія; Том 18 (2014): Сільськогосподарська мікробіологія; 132-145 1997-3004 10.35868/1997-3004.18 en uk https://smic.in.ua/index.php/journal/article/view/190/263 https://smic.in.ua/index.php/journal/article/view/190/264 Авторське право (c) 2014 Yu. P. Rud https://creativecommons.org/licenses/by/4.0 |
| spellingShingle | флавобактеріоз райдужна форель ПЛР Рудь, Ю. П. ЕКСПРЕС ДІАГНОСТИКА ФЛАВОБАКТЕРІОЗУ РИБ МЕТОДОМ ПОЛІМЕРАЗНОЇ ЛАНЦЮГОВОЇ РЕАКЦІЇ |
| title | ЕКСПРЕС ДІАГНОСТИКА ФЛАВОБАКТЕРІОЗУ РИБ МЕТОДОМ ПОЛІМЕРАЗНОЇ ЛАНЦЮГОВОЇ РЕАКЦІЇ |
| title_alt | RAPID DIAGNOSTIC OF FISH FLAVOBACTERIOSIS USING POLYMERASE CHAIN REACTION |
| title_full | ЕКСПРЕС ДІАГНОСТИКА ФЛАВОБАКТЕРІОЗУ РИБ МЕТОДОМ ПОЛІМЕРАЗНОЇ ЛАНЦЮГОВОЇ РЕАКЦІЇ |
| title_fullStr | ЕКСПРЕС ДІАГНОСТИКА ФЛАВОБАКТЕРІОЗУ РИБ МЕТОДОМ ПОЛІМЕРАЗНОЇ ЛАНЦЮГОВОЇ РЕАКЦІЇ |
| title_full_unstemmed | ЕКСПРЕС ДІАГНОСТИКА ФЛАВОБАКТЕРІОЗУ РИБ МЕТОДОМ ПОЛІМЕРАЗНОЇ ЛАНЦЮГОВОЇ РЕАКЦІЇ |
| title_short | ЕКСПРЕС ДІАГНОСТИКА ФЛАВОБАКТЕРІОЗУ РИБ МЕТОДОМ ПОЛІМЕРАЗНОЇ ЛАНЦЮГОВОЇ РЕАКЦІЇ |
| title_sort | експрес діагностика флавобактеріозу риб методом полімеразної ланцюгової реакції |
| topic | флавобактеріоз райдужна форель ПЛР |
| topic_facet | Flavobacteriosis rainbow trout PCR флавобактеріоз райдужна форель ПЛР |
| url | https://smic.in.ua/index.php/journal/article/view/190 |
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