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Pathogenic bacteria prevalence in cultured Nile tilapia in Southwest Mexico: A

real-time PCR analysis

Article in Journal of Fish Diseases · January 2024

DOI: 10.1111/jfd.13921

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Received: 28 September 2023 | Revised: 20 December 2023 | Accepted: 23 December 2023

DOI: 10.1111/jfd.13921

RESEARCH ARTICLE

Pathogenic bacteria prevalence in cultured Nile tilapia in

Southwest Mexico: A real-­time PCR analysis

Sonia A. Soto-­Rodriguez1 | Francis I. Marrujo Lopez1 | Karla G. Aguilar-­Rendon1 |

Rafael Hernández Guzmán2

1
Centro de Investigación en Alimentación
y Desarrollo, A.C. Coordinación Mazatlán, Abstract
Mazatlan, Sinaloa, Mexico
The present study investigates molecular-­based PCR techniques to estimate the prev-
2
Investigador por México CONAHCYT,
Universidad Michoacana de San Nicolás
alence of fish pathogens in southwest Mexico where recurrent mortality in the tilapia
de Hidalgo, Morelia, Michoacán, Mexico cultures has been observed. Sample of internal organs and lesions of Nile tilapia were

Correspondence
taken and analysed in 2018, 2019, 2020 and 2022 to detect bacterial pathogens using
Sonia A. Soto-­Rodriguez, Centro PCR. No samples were taken in 2021 due to the COVID-­19 pandemic. The real-­time
de Investigación en Alimentación
y Desarrollo, Unidad Mazatlán en
PCR conditions were optimized to allow a qualitative reliable detection of the bacteria
Acuicultura y Manejo Ambiental, Av. from fixed fish tissue. A total of 599 pond-­ and cage-­cultured tilapia from the south-
Sábalo Cerritos, Mazatlán, Sinaloa,
México.
western Mexican Pacific (Guerrero, Oaxaca and Chiapas states) were analysed. In
Email: ssoto@ciad.mx this tropical region, during 2018 and 2019 water temperatures of the tilapia cultures

Funding information
CONAHCYT grant FORDECyT 292474
“Sustainable regional development in the
South Pacific states of Mexico: a public
policy model for food sustainability
focused on tilapia farming in micro, small
and medium-­sized MIPYME enterprises
were generally with the optimal range to grow Nile tilapia, although extreme values
were recorded on some farms. Most of the tilapia sampled were apparently healthy.
No Francisella sp. was detected in any sample, and Staphylococcus sp. was the most
prevalent (from 0% to 64%) bacteria from the three states over time. Low prevalence
of Aeromonas sp. was found, from 0% to 4.3%, although the fish pathogen Aeromonas
dhakensis was not detected. Sterptococcus iniae was only detected in Chiapas in 2019
at a low prevalence (1.4%), while the major tilapia pathogen S. agalactiae was detected
at a high prevalence (from 0% to 59%) in the three Mexican states. This is the first
detection of these pathogenic bacteria in rural farms using real-­time PCR and consti-
tutes a great risk for tilapia aquaculture in Mexico, as well as a potential dispersion of
these pathogens to other aquaculture areas.

KEYWORDS
clinical signs, emerging fish pathogens, real-­time PCR, tropical region

1 | I NTRO D U C TI O N
Nile tilapia (Oreochromis niloticus), which is native to northern
Africa, is currently the third most abundantly farmed finfish
species worldwide, with commercial production recorded in 74
countries. The largest producer is China (1,241,410 t), followed by
Indonesia (1,172,633 t), Egypt (954,154 t), Brazil (343,596 t) and
Thailand (205,971 t) (FAO, 2022). World tilapia aquaculture pro-
duction grew 10.4% per year, from around 380,000 tons in 1990
to 6 million tons in 2018 (FAO, 2020). Thus, tilapia is not only an
important food source worldwide but is also one of global eco-
nomic importance. In general, tilapias are characterized by rapid
growth, they are very resistant to low oxygen levels (below 4 mg
O2 L−1) and high concentrations of organic matter in the water
(Arguedas et al., 2017) and are able to survive in large variations
of salinity and temperature (Fajer-­Ávila et al., 2017). Mexico is
currently the 9th largest producer of tilapia in the world, which
represents a food solution for the population from southwestern

J Fish Dis. 2024;00:e13921. wileyonlinelibrary.com/journal/jfd © 2024 John Wiley & Sons Ltd. | 1 of 11
https://doi.org/10.1111/jfd.13921
2 of 11 | SOTO-­RODRIGUEZ et al.
Mexico (CONAPESCA, 2017). However, as in any other farming
industry, tilapia is not exempt from diseases associated with ecto-
parasites, bacteria, viruses and fungi, although, most of research
have focused on parasitic diseases (García-­Vásquez et al., 2021;
Paredes-­
Trujillo et al., 2021). Cultured tilapia is susceptible to
bacterial pathogens distributed throughout tropical and temper-
ate regions where Nile tilapias are commonly cultured. In this
study, pathogens were selected due to their risk of outbreaks in
tilapias culture in Mexico. Francisella noatunensis subsp. orienta-
lis is an emerging pathogen of cultured tilapia around the world
causing a granulomatous disease (Leal et al., 2014; Nguyen
et al., 2016). In Mexico, it has also been identified as a pathogen
for cultured tilapia (Ortega Asencios et al., 2016). The significance
of Staphylococcus aureus is attributed to the rapid emergence
of antibiotic resistance among most of its isolates. Methicillin-­
resistant S. aureus has been related to mortality in Nile tilapia cul-
tures in northern Egypt (Soliman et al., 2014) and Malaysia (Atyah
et al., 2010). Recently, Staphylococcus haemolyticus was isolated
from tilapia farms in Chiapas, Mexico (Rajme-­Manzur et al., 2023).
Cultured tilapias are also susceptible to aeromonads causing
Motile Aeromonas Septicemia including Aeromonas sobria (Li &
Cai, 2011), Aeromonas veronii (Dong et al., 2015) and Aeromonas
dhakensis, the last of which was reported as a tilapia pathogen in
Mexico (Soto-­Rodriguez et al., 2018). Cocci Gram-­p ositive bacte-
ria Streptococcus iniae and Streptococcus agalactiae, also known as
Group B Streptococcus, have represented more than 90% of the
pathogens isolated from farmed dead tilapia each year resulting
in a mortality rate of 30%–90% (Liu et al., 2019). High prevalence
of both pathogens has been observed in tilapia cultured at over
26°C (Anshary et al., 2014). The present study was carried out in
a tropical region in southwestern Mexico (Guerrero, Oaxaca and
Chiapas states), reaching maximum water temperatures above
30°C, which favours periodic cases of streptococcosis, accom-
panied by lesions and mortality in adult animals close to harvest.
S. iniae and S. agalactiae have been identified in cultured tilapia
in different regions of Mexico, especially in hot humid climates
(Castro Ortiz, 2020; Hernandez et al., 2023; Ortega et al., 2018;
SAGARPA, 2022). Identification of fish pathogens around the
world is commonly based on biochemical and molecular tech-
niques. Molecular identification of pathogens results in rapid
detection to adopt preventive disease control strategies. Tilapia
from southwestern Mexico is currently cultured in small earthen
ponds or floating cages located in dams or small freshwater res-
ervoirs, seeded at low densities of fry in semi-­intensive systems.
Recurrent mortalities, associated with poor management and null
implementation of biosecurity measures during cultivation, have
been observed in past years in these regions. However, there are
no systematic health-­m onitoring programmes in place. In this con-
text, the aim of this study was to execute a health programme to
detect bacterial pathogens in samples of farmed tilapia from rural
farms using molecular techniques based on PCR. Real-­t ime PCR is
now a well-­e stablished method for the detection, quantification
and typing of different microbial agents and represents a powerful
tool in microbial diagnostics. In 2018, 2019, 2020 and 2022 sam-
ples of tilapia were taken directly by farmers and delivered to the
Bacteriology Laboratory to be analysed using conventional PCR
and real-­t ime PCR assays to detect bacterial pathogens. In some
years, the physicochemical parameters of ponds were registered
during the collection of the organisms.
2 | M ATE R I A L S A N D M E TH O DS
2.1 | Study area
This study was a health-­monitoring programme to detect fish patho-
gens in rural farms in Mexico. Tilapia farms, which consist of rustic
earthen ponds without aeration or floating cages inside dams in the
southwest of the Mexican Pacific Ocean (Figure 1) belonging to the
Guerrero, Oaxaca and Chiapas states. This Mexican region is mainly
characterized by a tropical wet and dry climate (Aw—Tropical, savan-
nah and Am—Tropical, monsoon), followed by a temperate climate
(Cwb—Temperate, dry winter, warm summer) (Beck et al., 2018). The
average annual temperature varies depending on the state, with 25,
22 and 23°C for Guerrero, Oaxaca and Chiapas, respectively. The
rains occur in summer from June to October with annual average
rainfall also varying from 1200 mm in the driest region (Guerrero)
to 1200–4000 mm in the wettest (Chiapas) per year (INEGI, 2023).
In 2018, 2019, 2020 and 2022, samples of Nile tilapia O. niloticus
(Linnaeus, 1758) were taken to be analysed. No samples were taken
in 2021 due to the COVID-­19 pandemic.
2.2 | Collection of organisms
In 2018, 2019 and 2020 field technicians of the State Aquaculture
Health Committees from Guerrero, Oaxaca and Chiapas states took
samples from each farm, while in 2022 samples were taken by tilapia
farmers. 5,6 tilapias/farm were collected in order to perform analy-
ses corresponding to the molecular identification of pathogenic bac-
teria (Table 1).
During the sampling, fish were obtained at random directly from
the ponds or cages and killed by a puncture in the head to be dis-
sected (OIE, 2022). Each tilapia was weighed and measured, and ex-
ternal observations such as skin colour, injuries, desquamation and
swelling were conducted for each farm. Internally, the aspect, colour
and contents of the body cavity were also reviewed. Then, samples for
bacterial detection were taken from adult and juvenile organisms from
the external skin lesions and the kidney, spleen, liver and brain were
individually dissected. For fry fish, the internal organs were aseptically
dissected and mixed with the brain. Tissue samples were fixed in eth-
anol at 96° and transported to the Laboratory of Bacteriology at CIAD
Mazatlan, Mexico. Water physicochemical parameters were measured
in 2018 and 2019 in the culture ponds or floating cages such as oxy-
gen (mg L−1), temperature (°C) and pH. Unfortunately, measurements
were not taken in 2020 or 2022.
SOTO-­RODRIGUEZ et al. | 3 of 11

F I G U R E 1 Location of the three southwest Pacific states of Mexico where tilapias (Oreochromis niloticus) were collected.

TA B L E 1 Number of tilapia samples analysed by site and year. strains used as positive controls was extracted using the Wizard®
Genomic DNA Purification Kit (Promega, Madison, WI, USA) accord-
No. No. samples
Year State No. farms fish analysed ing to the manufacturer's instructions. The obtained DNA was stored
at −20°C until PCR assay. DNA was quantified using a DeNovix DS-­
2018 Guerrero 14 70 280
11 Series (Thermo Fisher NanoDrop™ One Spectrophotometer).
Oaxaca 12 60 240
DNA was analysed for one-­step or real-­time PCR amplifications for
Chiapas 12 60 240
the pathogens Francisella sp., Staphylococcus sp., Staphylococcus au-
2019 Guerrero 12 67 268
reum, Aeromonas sp., Aeromonas dhakensis, Streptococcus iniae and
Oaxaca 12 61 244 Streptococcus agalactiae (Table 2). Strains used as positive controls
Chiapas 12 72 288 were stored at −80°C until use.
2020 Guerrero ns ns ns
Oaxaca 6 36 144
Chiapas ns ns ns 2.4 | Detection of Francisella using one-­step PCR
2022 Guerrero 6 37 148
Oaxaca 9 54 216 To confirm the Francisella genus, amplification was carried out

Chiapas 14 82 328
in a total reaction volume of 11.2 μL, composed of 3.0 μL PCR
Master Mix (Thermo Scientific™), 6.0 μL 18 Ohms deionized
Total 109 599 2396
water, (0.6 μL × 2) (0.1 mM) of each primer set (Table 2) and 1.0 μL
Abbreviation: ns, no sample.
(25 ng μL−1) of DNA template. Amplification was performed with a
SimpliAmp Thermal Cycler (Applied Biosystems®) using the fol-
2.3 | PCR and real-­time PCR assays lowing settings: initial denaturalization at 94°C for 30 s, followed
by 35 cycles at 94°C for 30 s, 60°C for 1 min and 72°C for1 min;
A total of 2396 samples from tilapia tissues were analysed. Genomic with a final extension at 72°C for 2 min. Genomic DNA from a F.
DNA from samples of kidney, spleen, liver and brain was extracted noatunensis subsp. orientalis strain, kindly donated by Dr. Esteban
using the cetyl trimethylammonium bromide (CTAB) buffer method Soto (The University of California), was included as a positive con-
modified by Doyle and Doyle (1987). DNA integrity was evaluated trol for each PCR assay. The negative control consisted of the same
by 1% agarose gel electrophoresis. Genomic DNA from the bacterial reaction mixture but without DNA. PCR amplification products
 4 of 11 | SOTO-­RODRIGUEZ et al.

were electrophoresed in 1.0% agarose gel at 120 V for 30 min at

Martínez-­Murcia et al. (2011)

Drancourt and Raoult (2002)

room temperature. A 100–3000 bp DNA ladder (AXYGEN) was
Forsman et al. (1994) used as a molecular marker. Gels were visualized under Axygen®
Gel Documentation Systems (Corning).

Atyah et al. (2010)

Zhou et al. (2011)

Cao et al. (2022)

Reference

2.5 | Standardization of primers for use in real-­time

PCR assay

Real-­t ime PCR was used as a highly reliable test to detect specific
82.0–82.5

85.5–86.0

78.0–78.5
86.5–87.0

bacteria in tilapia tissues, not to quantify bacteria, and, therefore,

Tm (°C)
a
1100

each set of primers was standardized before being used in the

88.0

assay. Although the primers used in this study were derived from
conventional PCR, they were selected for their high specificity to
discriminate bacteria at the species level. All primers were first
ACG TCA GAT GTG CAC AGT TAC TTA
GAAAA​TAG​GAA ​AGA​GAC​GCA​GTGTC
CCTTA​T TT​CCA​GTC​T TT​CGA​CCTTC

optimized to be used in real-­t ime PCR assays. Each set of prim-

GAG TTT GAT CCT GGC TCA GGA

ers (Table 1) was analysed by a temperature thermal gradient test

TAGCT ​TAG​T TA​TCC​C AA ​ATCCC
TACCA​GTT​GGA​A AC​GACTGT

GCI ACI TGI TCC ATA CCT GT

TAAAG​ACT​TCA​T TG​CGTGCC
CGTCA ​ATT​CCG​CCT​GATGCC
ATGCT​C AT​GCG​RCG​GTTGAT
AACCA ​ATT​CCG​TAT​IGGTTT

in order to establish the optimal hybridization temperatures. For

CCTTT​T TG​AGT​T TC​GCTCC

S. agalactiae the test temperatures were 56.0, 56.6, 57.8, 59.6,

61.7, 63.5, 64.5 and 65.0°C; for the rest of the bacteria they were
58.0, 58.7, 60.0, 62.0, 64.4, 66.4, 67.5 and 68.0°C. Amplifications
were performed with a SimpliAmp Thermal Cycler (Applied
Sequence

Biosystems®) using the following settings: initial denaturalization

1 cycle at 95°C for 3 min, followed by 34 cycles at 95°C for 20 s in
a gradient from 56.0 to 68.0°C, 30 s at 72°C, with a final exten-
sion from 65°C at 95°C every 5 s at 5°C. Melt Curve and Melt peak
analyses were also performed to evaluate the specificity of the
TA B L E 2 Bacterial strains used as a positive control for PCR detection of bacterial pathogens.

Sta3241R
Sta2491F

PCR product for detection of each pathogen using a CFX96 Touch

Primer

5058R
5057F
Saur R
Saur F

SP2
SP1
11R
F11

Real-­T ime PCR Detection System (BioRad). All protocols for the
8F
F5

identification of pathogens were modified according to the opti-

mal hybridization temperature.
Genus Aeromonas (rpoD RNA)
Genus Francisella (16S rRNA)

Genus Staphylococcus (16S)

Genus Staphylococcus (rpo)

S. iniae 16S-­23S rDNA

2.6 | Detection of bacterial pathogens using

real-­time PCR
GBS (cfb gene)
Specificity

Primers used for detection of Staphylococcus sp., S. aureus, Aeromonas

sp., A. dhakensis, S. iniae and S. agalactiae were synthesized in
T4OLIGO (Mexico). Amplification was carried out in a total reaction
volume of 5.0 μL, composed of 2.5 μL Luna® Universal qPCR Master
Amplicon size in bp; GBS: Group B Streptococcus.

Mix (New England Biolabs, UK), 1.8 μL 18 Ohms deionized water,

Streptococcus agalactiae ASG-­2290, AGS-­2292
Streptococcus iniae ATCC 29178 (CAIM 527 T )

(0.1 μL × 2) (0.1 mM) of each set of primers and 0.5 μL (25 ng μL−1) of
DNA template. Strains used as positive controls and sets of primers
Staphylococcus gallinarum CAIM 659

Staphylococcus gallinarum CAIM 659

Francisella noatunensis sub. orientalis

are in Table 2. The negative control consisted of the same reaction

Aeromonas dhakensis CAIM 1873

mixture without DNA. Two positive controls (genomic DNA from the
bacterial strain) and two negative controls (without genomic DNA)
were added to each reaction.
The real-­time PCR protocol for Staphylococcus sp. and S. aureus
consisted of 1 cycle of 30 s at 94°C, followed by 35 cycles of 30 s at
94°C, 30 s at 55°C and 1 min at 72°C; and the final extension was
from 65 to 95°C every 5 s at 5°C. The real-­time PCR procedure for
Strain

Aeromonas sp. consisted of 1 cycle of 30 s at 94°C, followed by 35 cy-

cles of 30 s at 94°C, 30 s at 55°C and 1 min at 72°C, and the final
a
SOTO-­RODRIGUEZ et al. | 5 of 11

extension was from 65 to 95°C every 5 s at 5°C. The PCR scheme for
S. iniae consisted of 1 cycle of 30 s at 94°C, followed by 35 cycles of
1 min at 94°C, 1 min at 60°C, and 1 min at 72°C, and the final exten-
sion was from 65 to 95°C every 5 s at 5°C. The PCR protocol for S.
agalactiae consisted of 1 cycle of 1 min at 94°C, followed by 35 cycles
of 15 s at 95°C, 30 s at 56.6°C, and the final extension was from 65
to 95°C every 5 s at 5°C. After the amplification of each pathogenic,
the melting step was performed to evaluate the Tm.
Detection of S. iniae showed two amplification temperatures
(77.0–77.5 and 85.5–86.0°C); therefore, to check the specificity of
the real-­time PCR assay for detecting S. iniae, the amplification tem-
perature was validated by one-­step PCR using the same protocol pre-
viously described including the reference strains S. iniae CAIM 527 T
(ATCC 29178) and S. iniae (R2845) isolated from O. niloticus farmed
in Mexico kindly donated by Dr. Gustavo Ramirez (CEVA United
Kingdom). The PCR and real-­time PCR amplification products were
electrophoresed in 1.0% agarose gel at 120 V for 30 min at room tem-
perature and analysed under a ChemiDoc XRS + Gel Imaging System
(BioRad).
Prevalence of pathogenic bacteria over time and origin were esti-
mated using the online programme Win Epi (Working in Epidemiology
http://​www.​winepi.​net/​uk/​index.​htm) with a 95% confidence level.
3 | R E S U LT S
3.1 | Clinical signs of collected tilapias
In 2018, most sampled tilapias from Guerrero, Oaxaca and Chiapas
were apparently healthy; some organisms showed eroded fins, white
cysts on fins, injured eyes, swollen kidneys, liver and spleen, ex-
ophthalmia, friable liver, ulcers, external lesions and desquamation
(Figure 2). In 2019, in Guerrero and Oaxaca, most organisms were
apparently healthy, although some tilapias displayed haemorrhagic
areas, pale and swollen livers, exophthalmia, swollen spleens and
petechiae in the operculum. However, half the tilapias from Chiapas
showed pale livers and a few organisms had swollen livers and des-
quamation. In 2020 there was no data on clinical signs. In 2022 the
clinical signs recorded from some organisms presented eroded fins,
swollen abdomens, injured skin, exophthalmia and desquamation.
3.2 | Physicochemical parameters
In 2018 the water temperature ranged from 21 to 32°C, while in
2019 it was slightly higher from 24 to 33°C (see Table S1). Dissolved

F I G U R E 2 External and internal

lesions observed on farmed Nile tilapia
(Oreochromis niloticus) from Guerrero,
Oaxaca and Chiapas. (a) Ulcers on
mandibula; (b) lesions on the head and
mouth; (c) cyst on pectoral fin; (d) pale
and swollen liver, white granules in spleen
and cysts on skin; (e) haemorrhage and
necrotic lesions on skeletal muscle; (f)
haemorrhage on skin.
6 of 11 | SOTO-­RODRIGUEZ et al.
oxygen in 2018 was highly variable, from 2.8 to 11.0 mg L−1, while in
−1
2019 it was from 2.3 to 10.0 mg L . In 2018 and 2019, values of pH
ranged from 7.0 to 9.0.
3.3 | Detection of Francisella sp.
No individuals of the Francisella genus were detected in all samples
of tilapia from Guerrero, Oaxaca and Chiapas in the time period
sampled using the one-­step PCR technique (see Figure S1). Figure S1
shows the amplicon (1100 bp) of the positive control for detection
of Francisella sp. and negative tissue samples of the internal organs
without amplification from Chiapas collected in 2019.
3.4 | Standardization of primers for real-­time PCR
Each set of primers was first standardized before being used to de-
tect the bacterial pathogens. Figures S2 and S3 show the graphs of
the amplification temperatures (°C) for the Melt Curve and Melt
Peak obtained for each set of primers for each pathogen, which were
described in the Materials and Methods section. The size of the am-
plicon of both S. iniae reference strains was similar (average 230 bp)
in both assays’ PCR and real-time PCR (Figure 3), which validate the
specificity of the set of primers used in this study.
The Ct/Cq (threshold/quantification cycle) values are the
numbers at which a sample's reaction crosses a fluorescence
threshold, indicating the detection of the target nucleic acid.
Ct values can be used for qualitative assays, such as determin-
ing the presence or absence of a pathogen. The mean Cq's for
the positive controls were 14.2 for Aeromonas dhakensis CAIM
1873, 17.01 for Staphylococcus gallinarum CAIM 659, 9.75 for
Streptococcus iniae CAIMN 527 T and 12.86 for Streptococcus aga-
lactiae ASG2290 and ASG2292. The ranges of these Cq values
are in Table S2.
3.5 | Detection of bacterial pathogens
Staphylococcus sp. was the most detected bacteria from the three
states over time; Aeromonas sp. was detected at low prevalence in
2018 in samples collected from Guerrero (4.3%) and Chiapas (1.7%),
however, the pathogen A. dhakensis was not detected from internal or-
gans of tilapia (Figure 4). S. iniae was only detected at a low prevalence
(1.4%) in Chiapas in 2019. S. agalactiae, which is highly pathogenic for
Nile tilapia, was detected with different prevalences in 2018, 2019 and
2022, with the highest prevalence (59%) in 2022 in Guerrero. Table S3
shows the prevalence and confidence interval of the analysed bacteria.
The range of Cq values for the above examples varied depending
on the bacteria. For Staphylococcus sp. it was from 25.92 to 30.83, for
Aeromonas sp. it was 25.73, for S. iniae it ranged from 20.55 to 23.56
and for S. agalactiae it ranged from 23.78 to 34.79. Figure 5 shows
examples of positive detection of Staphylococcus sp., Aeromonas sp.,
S. iniae, and S. agalactiae from internal organs of farmed O. niloticus.
An unspecific amplification at 77.0–77.5°C was observed in the S.
iniae real-time PCR assay, which was also observed during the stan-
dardization of the set of primers (Figure S3b).
4 | DISCUSSION
In this multi-­year study (2018, 2019, 2020 and 2022), samples of cul-
tured tilapia were analysed to detect fish pathogens using real-­time
PCR assays. Newly emerging or re-­emergent bacterial fish diseases,
such as francisellosis in the freshwater fish Micropterus salmoides
(Sheehy et al., 2023) and the cichlid Herichthys cyanoguttatus (Chang
et al., 2021), streptococcosis in tilapia (Cao et al., 2022; Zhang, 2021)
and aeromoniasis in tilapia (Azzam-­Sayuti et al., 2021) have been a
new threat for aquaculture, which may be attributed to close contact
of the aquaculture environment with animal and/or human wastes.
From 2018 to 2020, most of the sampled tilapias from Guerrero,
Oaxaca and Chiapas were apparently healthy. However, swollen livers
F I G U R E 3 Amplicons from PCR
and real-­time PCR products of S. iniae
strains. Lane M, 100 bp DNA ladder;
lines 1, 2, 5 and 6, S. iniae Si R2845 strain
(222–239 bp); lines 3, 4, 7 and 8, S. iniae
CAIM527 T (ATCC 29178) (223–237 bp);
NC, negative control.
SOTO-­RODRIGUEZ et al. | 7 of 11

F I G U R E 4 Prevalence of pathogenic bacteria detected in farmed Nile tilapia (O. niloticus) over time in Guerrero, Oaxaca and Chiapas
southwest Mexico. ns, no sample.

F I G U R E 5 Examples of positive detection (black lines), negative detection (grey lines) and positive control (blue lines) from tissue samples
of O. niloticus. (a) Detection of Staphylococcus sp. in Oaxaca and Chiapas collected in 2022. Positive control (Tm 82.0°C) of Staphylococcus
gallinarum CAIM 659; (b) detection of Aeromonas sp. in Chiapas collected in 2018. Positive control (Tm 88.0°C) of Aeromonas dhakensis CAIM
1873; (c) detection of S. iniae in Chiapas collected in 2019. Positive control (85.5°C) of Streptococcus iniae ATCC 29178; (d) detection of
Streptococcus agalactiae in Oaxaca collected in 2022. Positive control of S. agalactiae ASG 2290 (78.0–78.5°C). Negative controls (red lines).
8 of 11 | SOTO-­RODRIGUEZ et al.
and spleens were the most frequent clinical signs, which were also
observed in apparently healthy tilapias cultured in dams from Sinaloa
state, located in north-­western Mexico (Soto-­Rodriguez et al., 2013).
In this study, in 2022, most data were missing, although, some organ-
isms displayed swollen abdomens resulting from ascites, injured skin,
exophthalmia and desquamation, signs commonly associated with
Streptococcus infection in Nile tilapia (Amal & Zamri-­Saad, 2011).
Diagnosis of bacterial diseases commonly includes clinical signs,
wet mount preparations, necropsy, histopathology and molecu-
lar techniques. Molecular identification using PCR techniques for
potential pathogens is the key to early detection of asymptomatic
carrier fish. The commonly used molecular methods for detection
or identification include amplification of housekeeping genes or
sequences by conventional or multiplex PCR assays and 16S rRNA
sequencing. Quantitative real-­time PCR (qPCR) is used for quanti-
fication of bacteria, which allows the determination of the absolute
quantity of target DNA in the sample according to a calibration
curve constructed of serially diluted standard samples with known
concentrations or copy numbers.
Francisella sp. is detected using genus-­specific primers target-
ing 16S rRNA sequences in a one-­step PCR (Forsman et al., 1994;
Nguyen et al., 2016; Ortega Asencios et al., 2016) as well as se-
quencing of the 16S rRNA gene (Ramírez-­Paredes et al., 2017). In
this study, no Francisella sp. was detected using amplification of the
16S rRNA gene by conventional PCR.
Staphylococcus sp. was the most prevalent bacteria in the three
states over time. S. aureus was reported in O. niloticus, triggering high
mortality with various pathological alterations (Gaafar et al., 2015).
Methicillin-­resistant S. aureus has been detected by amplification
of mecA and mecC genes in conventional PCR tests (Elsayed Naeim
et al., 2023). Recently, Staphylococcus haemolyticus caused moderate
mortality in tilapia farms in Chiapas, Mexico and was identified by
amplification of 16S rRNA (Rajme-­Manzur et al., 2023); in contrast,
we used this gene in real-­time-­PCR assays.
Motile Aeromonas septicemia comprises several species that
possess virulence factors. A. dhakensis has been misidentified as A.
hydrophila, A. veronii or A. caviae; therefore, the importance that is at-
tributed to this bacterium in fish infections should be re-­evaluated due
to the changing taxonomy (Soto-­Rodriguez et al., 2018). Amplification
of 16S rRNA, gyrB, rpoB and rpoD genes using specific primers have
been commonly used for Aermonas sp. detection (Assane et al., 2021;
Korczak et al., 2004; Pridgeon & Klesius, 2011). In this study,
Aeromonas sp. was detected at low prevalence in 2018 in Guerrero
and Chiapas using the rpoD gene, but not the fish pathogen A. dhaken-
sis. Soto-­Rodriguez (2009) reported a 30% prevalence of presumably
A. hydrophila (identified using biochemical protocols) in cage-­cultured
tilapia from Sinaloa, Mexico's dams. Bacteria was isolated from the liv-
ers, kidneys and brains of tilapias displaying desquamation, blindness,
protruding and opaque eyes, eroded caudal fins and, pale gills.
Identification of Streptococcus sp. using biochemical profiles is
time-­consuming and phenotypic similarities among S. iniae and an-
other Streptococcus spp. or Lactococcus spp. may lead to misidenti-
fication. Single and multiplex PCR tests using lctO gene, 16S rDNA
(Mata et al., 2004; Park et al., 2014), the ITS region (16S to 23S rDNA)
(Berridge et al., 2001; Cui et al., 2019; Roach et al., 2006; Zhou
et al., 2011) and quantitative PCR (qPCR) (Phasunon et al., 2023) have
successfully been used to identify S. iniae. In this study, we used the
ITS region to detect S. iniae, however, the size of the amplicon of the
S. iniae CAIM 527T (ATCC 29178) reference strain was 230-­bp, in
contrast to 377-­bp reported by Zhou et al. (2011), probably due to our
modified amplification protocols. In this case, the specificity of prim-
ers was probed using two different PCR assays, one-­step and real-­
time PCR. S. iniae was detected at low prevalence in 2019 in Chiapas,
the Mexico's leading state in tilapia farming. The Mexican govern-
ment reported a high prevalence of S. iniae in Queretaro (29%) and
Guerrero (33%) in cultured tilapia (SAGARPA, 2022). Unfortunately,
the method used for bacterial identification was not mentioned. In
2017 Ortega et al. identified S. iniae by PCR and 16S rRNA sequencing
as the causative disease agent in two Mexican tilapia (Oreochromis au-
reus) populations. The identification of S. agalactiae in farmed tilapia is
critical for the welfare and management of diseases in populations of
fish. Thus, the development of a highly sensitive and rapid diagnostic
method is essential. 16S-­23S rDNA has been used for identification
by single PCR and real-­time PCR (Ortega Asencios et al., 2016; Cui
et al., 2019) as well as 16S rRNA (Piamsomboon et al., 2022). The
single copy cfb gene has been used for identification and quantifi-
cation of S. agalactiae, which encodes the CAMP factor, by real-­time
PCR (Cao et al., 2022; Sebastiao et al., 2015), which was also used
in this study for detection of the pathogen. The major tilapia patho-
gen S. agalactiae had high prevalence in 2022 in Guerrero, Oaxaca
and Chiapas (59%, 26% and 26%, respectively), which constitutes a
threat to the state's aquaculture. This difference in prevalence could
be due to several factors, such as the size, production system and
implementation of good practices of each farm. Cumulative mortal-
ity during the chronic phase in tilapia can reach 80% (FAO, 2021). In
the chronic form, light brown or dark red nodules were seen in the
skeletal muscle near the vertebra of Nile tilapia (Li et al., 2014) and
lesions were also observed in this study (Figure 2e). Streptococcosis
caused by S. agalactiae is highly contagious and is directly related to
high temperatures increasing their incidence and vulnerability to the
disease. Outbreaks caused by S. agalactiae infections in O. niloticus
farms in Brazil revealed high mortality and disease occurrence at
water temperatures above 26°C (Mian et al., 2009). Mortality in Nile
tilapia cultured in cages in Indonesia was registered at temperatures
over 28°C (Anshary et al., 2014).
Outbreaks in 2019 caused by S. agalactiae 1a in Mexico, caused
tilapia mortality of over 60% in vaccinated batches and up to 85% in
unvaccinated batches, at water temperatures over 32°C (The Fish
Health, 2019). The Mexican government reported 33% prevalence
of S. agalactiae in Oaxaca, 12% in Queretaro and, 20% in Veracruz
in cultured tilapia (SAGARPA, 2022), but again, the method used
for bacterial identification was missing. S. agalactiae and S. iniae
were identified in 2017 using 16S rRNA sequencing from cultured
tilapias from a Guerrero dam, Mexico (Castro Ortiz, 2020), but no
prevalence was reported. In Malpaso Dam located in Chiapas, 80%
of the tilapia farms with 50% mortality, were presumably positive
SOTO-­RODRIGUEZ et al.
for Streptococcus spp., using biochemical methods for identifica-
tion (Hernández Hernández et al., 2021). High temperatures and
low oxygen, along with rainy and dry periods, were considered the
main climate-­related risks in floating cages, that predispose stress
and susceptibility to streptococcosis (Cochrane et al., 2009; Lebel
et al., 2016).
In this study during 2018 and 2019 water temperatures of the ti-
lapia cultures were generally within the optimum range to grow Nile
tilapia, although high and low values were recorded on some farms.
The optimal temperature range, defined as the thermal range where
80% or more of the metabolic scope can be maintained, is between
19.5 and 32.1°C (Leonard & Skov, 2022), but no clear relation between
the physicochemical variables and pathogen presence was found due
to lack of data. In Mexico, tilapia mortality in rural farms is recurrent
every year, but information about infectious diseases is scarce; the
cause, the etiological agent or the predisposing condition is unknown,
attributing mortalities to poor water quality and inadequate culture
management. In this study, in the three states sampled over time, we
found from one to three bacterial pathogens in rural farms, which
means that these pathogens can cause a co-­infection and constitute a
big risk for aquaculture in Mexico. Future studies should focus on the
quantification of each pathogen and make multiple comparisons that
include region and time. Figure 5 shows examples of positive detec-
tion of Staphylococcus sp., Aeromonas sp., S. iniae and S. agalactiae
from internal organs of farmed O. niloticus. An unspecific amplifica-
tion at 77.0 –77.5 °C was observed in the S. iniae real-time PCR assay,
which was also observed during the standardization of the set of prim-
ers (Figure S3b).
5 | CO N C LU S I O N S
High prevalence of the potential fish pathogen Staphylococcus sp.
and the major tilapia pathogen S. agalactiae were found in the in-
ternal organs of farmed tilapia from Guerrero, Oaxaca and Chiapas,
tropical states located in the South Pacific Ocean, while the patho-
gens S. iniae and Aeromonas sp. were detected at low prevalence.
All pathogens were present in the three sampled states over time,
indicating a co-­infection process in farmed tilapia that represents a
major threat to sustainable fish farming in Mexico. Fortunately, the
other major pathogen Francisella sp. was not detected at any time.
No clear clinical signs were related to the bacterial pathogens de-
tected in farmed tilapia from the three Mexican states. Real-­time
PCR for bacterial detection is a fast and highly reliable test to iden-
tify bacterial fish pathogens in fixed tissues.
AU T H O R C O N T R I B U T I O N S
Sonia A. Soto-­Rodriguez: Conceptualization; investigation; funding
acquisition; writing – original draft; resources. Francis I. Marrujo
Lopez: Formal analysis; writing – review and editing; methodology.
Karla G. Aguilar-­Rendon: Writing – review and editing; formal analy-
sis; methodology. Rafael Hernández Guzmán: Formal analysis.
| 9 of 11
AC K N O​W L E D
​ G E ​M E N T S
The authors would like to thank the Committee of Aquaculture
Health of the State of Guerrero, Oaxaca Committee of Aquaculture
Health and Safety and the State Committee for Aquaculture Health
of Chiapas for their help during the collection of organisms. Thanks to
Julissa Enciso for her Technical support and Circe Romero for logistical
support in rural farms.
F U N D I N G I N FO R M AT I O N
The present study was supported by CONAHCYT grant FORDECyT
292474 ‘Sustainable regional development in the South Pacific states
of Mexico: a public policy model for food sustainability focused on
tilapia farming in micro, small and medium-­sized MIPYME enter-
prises’ (Desarrollo regional sustentable en los estados del Pacífico
Sur Mexicano: modelo de política pública para la sustentabilidad
alimentaria enfocado en el cultivo de tilapia en la micro, pequeña y
mediana MIPYME empresa).
C O N FL I C T O F I N T E R E S T S TAT E M E N T
The authors declare that they have no conflict of interest.
DATA AVA I L A B I L I T Y S TAT E M E N T
The manuscript does not contain shared data.
ORCID
Sonia A. Soto-­Rodriguez https://orcid.org/0000-0003-0760-4029
Rafael Hernández Guzmán https://orcid.org/0000-0002-2711-9015
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S U P P O R T I N G I N FO R M AT I O N
Additional supporting information can be found online in the
Supporting Information section at the end of this article.
How to cite this article: Soto-­Rodriguez, S. A., Marrujo Lopez,
F. I., Aguilar-­Rendon, K. G., & Guzmán, R. H. (2024).
Pathogenic bacteria prevalence in cultured Nile tilapia in
Southwest Mexico: A real-­time PCR analysis. Journal of Fish
Diseases, 00, e13921. https://doi.org/10.1111/jfd.13921