A review of molecular diagnoses of bacterial fish

diseases

https://doi.org/10.1007/s10499-022-00983-8
 Overview
Abstract

Mass mortalities in aquaculture

Bacterial coinfections

Mobile elements and virulence

Limitations of traditional techniques in bacterial identification

Molecular techniques for identifying fish pathogens

 Abstract
• World's 9th largest fish producer
Egypt • Total production 1.5 billion tons per year
• Almost 79.6% of farmed fishes

• Reported massive mortalities in marine

and freshwater fishes
• Resulted in serious economic
Losses in the country
 1. Poor biosecurity practices
Mortality 2. Lack of awareness among smallholder fish
Reasons?
farmers
3. Temperature fluctuations

post-mortem Septicemic
lesions clinical signs

Moribund fishes

Bacterial Disease outbreak Fungal

Pathogens Infection

Modern Molecular Techniques

 Mass mortalities in aquaculture
Unexpected, sudden, and Egypt has experienced
acute mass mortalities eruptions of mass kills
Fish production declined
among the wild and caused by various septicemic
from 82.9 to 67.3%
farmed fish population bacterial pathogens that
(Abdelsalam et al. 2021).
over a short time (El- involved cultured marine and
Mezayen et al. 2018) freshwater fishes

Egyptian government reveals

and finances several fish
farming projects and
encourages the private
sector to invest in
aquaculture
Rapid identification of
pathogens affecting
cultured fish (Eissa et al.
2021a).
 Bacterial Coinfection
Two or more genetically
It can significantly affect the dynamics
different microbial
of fish disease outbreaks by amplifying
pathogens infect the
the severity, duration, and progression
same host simultaneously
of infection

In synergistic In Antagonistic
coinfection, (Dong et Coinfection, (Kotab et
al. 2016). al, 1016)

Diminish the
Difficult to diagnose
vaccination efficiency
the coinfection
in moribund fish
(Carella et al. 2020)
(Figueroa et al. 2017)
 Mobile elements and virulence
Pathogenic pathways of septicemic bacterial infections are triggered by several
harmful extracellular products (ECPs) (Baffone et al. 2001)

Several virulence genes are frequently linked with mobile genetic elements,
such as insertion sequences (IS), promoting horizontal gene transfer.

IS plays a significant role in rearranging genetic materials such as deletion,

translocation, inversion, and duplication (Abdelsalam et al. 2009; 2010; 2015).

Mobile elements promote bacterial virulence.

 Limitations of traditional techniques in bacterial
identification
comprise colony shape, Gram stain, a motility test, catalase and/or oxidase reaction, and
oxidation/fermentation of glucose (Cai et al. 2014).

Proper selection of diseased tissue

Proper selection of specific and general media

Appropriate incubation conditions

Appropriate selection of meaningful colonies for further study (Austin, 2019).

Avoid the contamination

Difficult to identify the primary host in moribund fish

Methods are time-consuming

 Molecular techniques for identifying fish
pathogens
limitations of conventional methods have resulted in the expansion of the
usage of molecular techniques depending upon their higher specificity and
sensitivity.

• Polymerase chain reaction

• Nested PCR
• Multiplex PCR
• Real‑time PCR
• Loop‑mediated isothermal amplification
• Universal DNA sequencing
• DNA microarrays
 Polymerase chain reaction
• DNA-based diagnostic assay
• PCR technique can amplify millions of copies of specific gene
sequences from a small amount of DNA using specific primers
and DNA polymerase.

• PCR cycling includes three stages:

• 1. Denaturation: separation of the two strands of the DNA at
96 °C.
• 2. Annealing: Attachment of primers at specific regions on
DNA strands
• 3. Extension: DNA polymerase at 72 °C
PCR is usually carried out in 25–40 cycles and should yield the expected size
of the amplicons (Abdelsalam et al. 2017).

The size of amplicons could be detected through gel electrophoresis (Eissa

et al. 2021a, b).

In the field of diagnosis of fish bacterial diseases, this technique is used

for the detection of A. hydrophila, A. salmonicida, V. anguillarum, Y. ruckeri,
L. garvieae, P. damselae, and S. dysgalactiae (Abdelsalam et al. 2017; Eissa
et al. 2021a, b).
Nested PCR

For the sake of increasing Two sets of different In egypt, nested PCR has been
the sensitivity and primer pairs were successfully applied in the
specificity of the PCR applied in two diagnosis of mixed infection of
reaction successive PCR streptococcus and myxobolus
amplifications. tilapiae coinfections in moribund
nile tilapia (eissa et al. 2021b).
Multiplex PCR
• Multiplex PCR can simultaneously target and amplify several specific genes
from different bacterial pathogens using multiple primer pairs and yield
different sizes of specific DNA products.

• This method has been applied for concurrent identification of three

different pathogenic bacteria (A. hydrophila, F. columnare, and E. ictaluri),
five different pathogenic bacteria (Y. ruckeri, A. salmonicida subsp.
salmonicida, A. hydrophila, R. salmoninarum, and F. columnare ) (Farzadnia
and Naeemipour 2020), and four different pathogenic bacteria ( V.
parahaemolyticus, V. cholerae, V. alginolyticus, and V. vulnificus ) (Adams and
Thompson 2011).
Real-time PCR

The sensitivity and

The RT-PCR method is
specificity of real-time PCR
repeated 25–50 times and
are very high and can
involves a series of
determine very low levels of
temperature fluctuations
DNA in different tissue
(Austin 2019).
samples.

Real-time PCR is used for

the identification and The assay is an
quantification of pathogenic
bacteria in fish such as A.
expensive test and hard
salmonicida and S. to scale.
parauberis (Austin 2019).
Loop‑mediated isothermal amplification

• LAMP is a cost-effective, simple, fast, and highly specific

method for amplification of nucleic acid. This tool uses six
different primer sequences and a Bst DNA polymerase to
target specific genes. This polymerase enzyme has the strong
5′–3′ DNA polymerase Activity.

• LAMP techniques have been applied for the identification of

E. tarda, V. anguillarum, and S. iniae (Austin 2019).
Universal DNA sequencing

• Molecular identification of retrieved bacterial isolates was

carried out by targeting specific genes.

• Amplification of the 16S rRNA gene followed by sequencing

• The size of the 16S rRNA gene is 1500 bp which is considered

to be enough for bioinformatics resolutions (Azwai et al. 2016).
DNA microarrays

• identification of several microbes in a single hybridization

assay (Austin 2019).

• Microarrays are very sensitive and specific tools used for

concur- rent diagnosis of five different bacterial pathogens:
A. salmonicida, V. parahaemolyticus, V. anguillarum, V.
vulnificus, and P. damselae subsp. damselae.
Conclusion
Bacterial coinfections
are often recorded in
wild and farmed fish.
In mixed types of infection,
traditional methods only for
diagnosis and may mask the
identity of real causes.

On the other hand,

molecular methods The whole genome sequencing of
have improved the microorganisms has made
ability to identify significant progress in
bacterial pathogens. understanding pathogen biology.

These techniques also provide a way to

examine the connections between the
genotype and phenotype of different micro
agents.
Drawbacks in Review Article
• Authors only review the bacterial pathogenic literature.
• Gave more preference for Modern molecular techniques.