Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73: e20253577, enero-diciembre 2025 (Publicado Dic. 02, 2025)

Frequencies and factors associated with pirA, pirB

and Vibrio parahaemolyticus (Vibrionales: Vibrionaceae)

in shrimps and farm waters of Costa Rica

Andrea García-Quesada1*; https://orcid.org/0000-0001-7280-8146

Marianita Chavarría-Alvarado1; https://orcid.org/0009-0006-7676-2046

1. Laboratorio de Biotecnología, Recinto de Grecia, Sede de Occidente, Universidad de Costa Rica, Grecia, Alajuela,

Costa Rica; andrea.garcia@ucr.ac.cr (*Correspondencia), marianita.chavarria_a@ucr.ac.cr

Received 10-IX-2024. Corrected 20-III-2025. Accepted 24-XI-2025.

ABSTRACT

Introduction: Acute Hepatopancreatic Necrosis Disease (AHPND) has been reported in cultured shrimp in

recent years and is associated with the bacterium Vibrio parahaemolyticus, containing a plasmid with the pirA

and pirB genes that confer pathogenicity.

Objective: To establish the presence of pirA, pirB and V. parahaemolyticus genes in postlarvae, juvenile shrimp,

and water from farms in the dry and rainy seasons in Costa Rica, as well as to determine associated factors such

as season of the year, farm, organ, pond water with the pirA gene, with the pirB gene or with the tlh gene, pond

site, and time of shrimp collection.

Methods: Two shrimp farms in Costa Rica were studied in 2021. A total of 72 juvenile shrimp samples, four

groups of postlarvae, and 16 water samples were analyzed. The genes of pirA, pirB and V. parahaemolitycus (tlh

+) were identified by qPCR, and associated factors were determined.

Results: V. parahaemolitycus was identified in 33.3 % of the juvenile shrimp samples, in a group of postlarvae,

and in 50 % of the water samples. In the shrimp samples 43 and 45 % were positive for the pirA and pirB gene

amplicon, respectively. Of the water samples, 31.2 and 37.5 % were positive for the pirA and pirB gene ampli-

con, respectively. Two factors associated with V. parahaemolitycus bacteria (rainy season and tlh-positive water),

and five factors associated with pirA and pirB genes (rainy season, presence of the tlh gene in water, pirA gene in

water, pirB gene in water, and time of collection) were found. Differences in salinity values were found between

dry and rainy season in water samples.

Conclusions: The studied genes were successfully detected in shrimp and pond water samples collected during

both seasons in Costa Rica. However, detection was more frequent during the rainy season.

Key words: AHNPD; pirA gene; Vibrio spp; diseases in shrimp; tlh gene.

RESUMEN

Frecuencias y factores asociados con pirA pirB y Vibrio parahaemolyticus

(Vibrionales: Vibrionaceae) en camarones y aguas de granjas en Costa Rica

Introducción: En los últimos años se ha reportado la Enfermedad de la Necrosis Aguda del Hepatopáncreas

(AHPND) en camarones de cultivo y se asocia a la bacteria Vibrio parahaemolyticus que contiene un plásmido

con los genes pirA y pirB que le confieren la patogenicidad.

Objetivo: Establecer la presencia de los genes pirA, pirB y de V. parahaemolyticus en postlarvas, en camarones

juveniles y agua de granjas en la estación seca y lluviosa en Costa Rica, así como determinar factores asociados

https://doi.org/10.15517/ac8dr232

GENETICS

2Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73: e20253577, enero-diciembre 2025 (Publicado Dic. 02, 2025)

INTRODUCTION

The increase in demand for food, as well

as the search for more nutritious, healthy and

sustainable diets by the wider public, have led

to the expansion of marine product consump-

tion in recent years. Seafood, being an excel-

lent source of minerals and vitamins, opens the

opportunity for the production of food under

the modality of aquaculture, as is the case of

shrimp farming. This activity has shown great

potential and growth both in the American con-

tinent, as well as in the rest of the world (Varela

& Varela-Moraga, 2019).

However, these farms reach a high density

of animals to obtain higher productive yields,

which causes a stressful environment for the

shrimp, providing ideal circumstances for dis-

ease outbreaks. As a result, this industry has

been impacted by the consequences of infec-

tious diseases of viral and bacterial origin,

effecting significant economic losses (Flegel,

2019; Martínez-Chávez et al., 2022).

On the other hand, environmental factors,

both biotic and abiotic, affect the immune

system of shrimp, with these effects being

more pronounced due to the use of high animal

densities. Abiotic factors include temperature,

salinity, concentration of nitrogen compounds,

pH and dissolved oxygen. However, in recent

years, there has been a growing interest in the

study of biotic factors such as the composition

of the intestinal microbiota, the presence of

biological production compounds by aquatic

microorganisms, the use of probiotics, prebiot-

ics and sustainable technologies such as biofloc

(Martín-Ríos et al., 2022).

In recent years, a disease affecting farmed

shrimp called Acute Hepatopancreatic Necrosis

Disease (AHPND) has been presented, which

was initially associated with a strain of the

bacterium Vibrio parahaemolyticus (Tran et

al., 2013). V. parahaemolyticus is a Gram-neg-

ative, halophytic bacterium that is widespread

in estuarine, marine and coastal environments

(Broberg et al., 2011; Ceccarelli et al., 2013;

Letchumanan et al., 2014), this bacterium pos-

sesses a plasmid containing two genes coding

for the synthesis of PirA and PirB toxins, which

confer pathogenicity (Han, Tang & Lightner,

2015). It is now known that other species of

the genus Vibrio have also been identified as

responsible for AHPND (Dong et al., 2017;

Kondo et al., 2015; Liu et al., 2018; Restrepo

et al., 2018).

In Costa Rica, there is a molecular study

whose objective was to investigate the presence

of Vibrio spp. and the plasmid genes encoding

PirA and PirB toxins in shrimp farms. The

study detected pirA and pirB genes in 33.3 %

(5 / 15) and Vibrio spp. in 40.0 % (6 / 15) of

the farms. In two of the six farms where Vibrio

como estación del año, finca, órgano, agua de estanque con el gen pirA, con el gen pirB o con el gen tlh, sitio del

estanque y tiempo de recolecta de los camarones.

Métodos: Se estudiaron dos fincas camaroneras en Costa Rica en el 2021. En total se analizaron 72 muestras de

camarones juveniles, cuatro grupos de postlarvas y 16 muestras de agua. Se identificaron los genes de pirA, pirB

y V. parahaemolitycus (tlh +) mediante qPCR y se determinaron factores asociados.

Resultados: Se identificó V. parahaemolitycus en el 33.3 % de las muestras de camarones juveniles, en un grupo

de postlarvas y en el 50.0 % de las muestras de aguas. En las muestras de camarones 43.0 y 45.0 % fueron posi-

tivas para el amplicón del gen pirA y pirB, respectivamente. Con respecto a las muestras de agua 31.2 y 37.5 %

resultaron positivas para el amplicón del gen pirA y pirB, respectivamente. Se encontraron dos factores asociados

a la bacteria V. parahaemolitycus (época lluviosa y agua positiva al gen tlh) y cinco factores asociados a los genes

pirA y pirB (época lluviosa, presencia del gen tlh en agua, gen pirA en agua, gen pirB en agua y tiempo de reco-

lecta). Se encontraron diferencias en los valores de salinidad entre la época seca y lluviosa en muestras de agua.

Conclusiones: Los genes estudiados fueron detectados con éxito en muestras de camarones y aguas de estanques

durante ambas estaciones del año. Sin embargo, la detección fue más frecuente durante el invierno.

Palabras clave: AHNPD; gen pirA; Vibrio spp.; enfermedades en camarón; gen tlh.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73: e20253577, enero-diciembre 2025 (Publicado Dic. 02, 2025)

spp. was found, the species V. parahaemolyti-

cus was identified (Peña-Navarro et al., 2020).

However, in Costa Rica there are no peri-

odic studies that evaluate the analyzed genes

through sampling at different points in time,

nor are there investigations that determine the

influence of the two seasons of the year on the

prevalence of the disease.

The objective of the present study was to

investigate the presence of pirA, pirB genes

and V. parahaemolyticus species in postlarvae

and juvenile shrimp of the species Litopeneaus

vannamei and in pond water samples, in two

farms located in the Gulf of Nicoya, Costa

Rica, in both dry and rainy seasons, as well as

to establish factors associated with pirA, pirB

genes and V. parahaemolyticus bacteria.

MATERIALS AND METHODS

Study design, origin and sample size: A

longitudinal non-experimental research design

of trend was carried out, given that the indepen-

dent variables were not intentionally altered,

but rather the phenomena were observed as they

occurred in the natural context. In addition, the

studies collected data at different points in time,

using a series of samples that covered different

participants at each moment, which were part

of the same population (Hernández-Sampieri et

al., 2014). Two private shrimp farms (Farm A

and Farm B) using the conventional production

method, located in the Gulf of Nicoya on the

Pacific coast of Costa Rica, were studied.

In Costa Rica, the aquaculture of white

shrimp of the genus Litopenaeus is primarily

developed in the Gulf of Nicoya, and the Cen-

tral and South Pacific areas (Instituto Costarri-

cense de Pesca y Acuicultura 2020). However,

the Gulf of Nicoya is the area of Costa Rica

with the greatest potential for the cultivation of

aquaculture species (Quesada-Céspedes et al.,

2019). It is characterized by a rainy period from

May to November (rainy season) and a period

where rainfall is scarce between December and

April (dry season) (Herrera, 2016).

During the harvest months, both farms

sampling was carried out approximately every

three to four weeks, starting with sampling one

(time 0, day of stocking), sampling two (three

to four weeks after stocking), sampling three

(six to seven weeks after stocking) and sam-

pling four (nine to ten weeks after stocking).

Dry season samples were collected between

February and April and rainy season samples

between July and September 2021. On each

farm, a pond of approximately four hectares

was sampled.

Both shrimp of the species Litopenaeus

vannamei (postlarvae and juveniles), and pond

water were studied. The postlarvae studied

were of national origin and came from a labora-

tory near the area, supplier of the two farms. A

total of 72 samples (36 hepatopancreas and 36

intestines) from 36 groups of juvenile shrimps

and four groups of postlarvae were analyzed.

Sampling of juvenile shrimp was done at three

points in the pond using cast nets: inlet, center

and outlet of the water, five shrimp were sam-

pled at each point, for a total of three groups

per farm per sampling, the three groups were

placed in different sterile containers with water

from the same pond.

Water sampling in the ponds was done at

only one point in the pond; in the central part,

one sample was taken per farm per sampling,

for a total of 16 water samples from the two

farms and from the two seasons of the year.

Approximately 250 ml of water were sampled

at 10 cm from the surface in sterile bags.

In each sampling, in situ physical chemical

parameters of the water were measured in the

center of the pond: temperature, pH, dissolved

oxygen and salinity, using a LabQuest 2 inter-

face and temperature, pH, dissolved oxygen

and salinity probes.

All samples were placed in a cooler and

transported to the Biotechnology Laboratory of

the Greece Campus of the University of Costa

Rica, where they were stored at 4 °C until anal-

ysis, no more than 24 hours after collection.

Bacterial strains and culture condi-

tions: The following strains were used as

positive controls: V. parahaemolyticus (pirA+

and pirB+, donated by the Universidad Técnica

4Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73: e20253577, enero-diciembre 2025 (Publicado Dic. 02, 2025)

Nacional, Sede Regional de Guanacaste, Costa

Rica) and V. parahaemolyticus ATCC 17802

(tlh+). Nuclease-free water was used as a

control without template and commercially

acquired Escherichia coli K-12 and a strain of

Vibrio alginolyticus, donated by the Faculty

of Microbiology of the University of Costa

Rica, were used as negative controls for the

genes studied.

The shrimp groups collected at each sam-

pling point were necropsied aseptically, the

hepatopancreas and intestine were extracted,

and then these organs were macerated separate-

ly. In the case of postlarvae, hundreds of them

were macerated. The macerates were enriched

in APW (alkaline peptonized water) with 2 %

sodium chloride.

The water samples were homogenized, and

25 ml of each sample was taken and enriched

separately in 225 ml of APW with 2 % sodium

chloride (Kaysner et al., 2004). The water

and shrimp samples in APW were then incu-

bated for six hours at 37 °C. Then they were

inoculated on TCBS agar (thiosulfate citrate

bile sucrose agar) with 1 µl, 10 µl and 100 µl

of each of the enriched samples, using sterile

micropipettes and Drigalsky loops. The inocu-

lated plates were placed in the incubator at a

temperature of 37 °C for 24 hours.

Extraction and verification of DNA

quality and quantity: DNA was extracted

from colonies grown on TCBS plates, com-

patible with morphological characteristics

reported for the species V. parahaemolyticus:

round, blue-green, opaque colonies, 2 to 3 mm

in diameter (Kaysner et al., 2004; Nelapati

et al., 2012). The extraction kit used was

Pure Link Genomic DNA Mini Kit Invitrogen

(K1820-01), the manufacturer’s protocol was

followed, with some modifications. Once the

DNA was extracted from the samples, it was

stored at -20 °C.

The quantity and quality of DNA in the

samples was verified by measuring absorbance

at 260 nm in a microvolume spectrophotometer

(NanoDrop One Thermo Fisher Scientific) and

by fluorescence (Qubit® 4 fluorimeter, Thermo

Fisher Scientific-Invitrogen). All samples had

the required amount of total DNA (on average

23 ng/µl) to be analyzed by qPCR. Samples

were run on 2 % agarose gels to confirm

their integrity.

Molecular identification: Identification

of V. parahaemolyticus bacteria species was

carried out by qPCR amplification of a partial

sequence of approximately 248 bp of the tlh

(thermolysin thermolabile hemolysin) gene fol-

lowing the protocol described by Micky et al.

(2014), with the following modifications: each

25 µL reaction was carried out with a final

concentration of 1X Maxima SYBR Green/

ROX qPCR Master Mix (2X) Thermo Scien-

tific and, as for the qPCR conditions an initial

denaturation for 7 min at 95 °C was used,

and a final extension of 72 °C for 7 min was

added (Table 1).

Identification of the amplicons of the pirA

and pirB genes was carried out by implement-

ing two different qPCR protocols, the primers

used were designed by Han, Tang, Pantoja et al.

(2015); Han, Tang, Tran et al. (2015), respec-

tively. A partial sequence of approximately 135

bp of the pirA gene was amplified, a reaction

was prepared with 10 µl of Maxima SYBR

Green/ROX qPCR Master Mix (2X) Thermo

Scientific, 2 µl of each primer (15 µM), 2 µl of

the extracted DNA and 4 µl molecular biology

grade water (Thermo Scientific), for a final

volume of 20 µl. The qPCR conditions used are

specified in Table 1.

For the partial sequence of the pirB gene, a

fragment of approximately 102 bp was ampli-

fied, a reaction was prepared with 10 µl of

Maxima SYBR Green/ROX qPCR Master Mix

(2X) Thermo Scientific, 2 µl of each primer

(3 000 nM), 1 µl of the extracted DNA and

5 µl of molecular biology grade water (Ther-

mo Scientific), for a final volume of 20 µl.

The qPCR conditions were the same as those

used for the amplification of the pirA gene

amplicon (Table 1).

The efficiency of the three protocols imple-

mented in qPCR was determined by standard

curves of 5 dilutions, each dilution in triplicate.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73: e20253577, enero-diciembre 2025 (Publicado Dic. 02, 2025)

The curves showed efficiencies of 100, 110 and

106 % for the partial sequence of the tlh, pirA

and pirB genes, respectively. The specificity

of the protocols was evaluated in two ways: 1)

qPCR products from the three standard curves

were run on a 1.5 % agarose gel to corrobo-

rate the existence of a single product with the

expected size, 2) dissociation curve analyses

were performed at the end of each PCR run,

which always showed a single amplified prod-

uct. Samples with Ct (threshold cycle) values

between 14 and 35 were considered positive.

Positive samples for pirA, pirB genes

were subjected to conventional PCR protocols

for confirmation and the protocol and primers

Samples that were successfully amplified by

conventional PCR for the pirA genes and some

samples amplified by qPCR for the tlh gene

were sent for sequencing to CIBCM (Centro de

Investigación en Biología Celular y Molecular)

at the University of Costa Rica. The sequences

of the samples were aligned with the BioEdit

Sequence Alligment Editor®2.2.28 program

and compared using the BLASTn® 2.2.28

algorithm with the NCBI database.

Statistical analysis: Factors associ-

ated with the pirA, pirB and tlh genes of

qPCR-positive shrimp were studied using a

non-conditional logistic regression model. The

risk was estimated using the odds ratio (OR)

and those factors with a p < 0.05 were consid-

ered associated factors. The factors analyzed

were season of the year, farm, organ, water

with the pirA gene, water with the pirB gene,

water with the tlh gene, pond site and time of

shrimp collection.

Depending on the distribution of the data,

an independent sample t-test was performed for

pH and dissolved oxygen and a nonparametric

Mann Whitney U test for temperature and

salinity to compare the averages or medians of

the physicochemical parameters between the

positive and negative water samples for the tlh,

pirA and pirB genes, in order to determine if

there are differences between the two groups.

Likewise, the averages or medians of the

physicochemical parameters obtained during

dry season and rainy season were obtained

and compared between them to determine

any existing differences. The Mann Whitney

U-test was used for samples with non-normal

distribution (temperature and oxygen) and the

T-test for samples with normal distribution

(salinity and pH).

Statistically significant differences were

considered for those values with a p < 0.05. All

Tab l e 1

Conditions used in the qPCR protocols for molecular identification of tlh, pirA and pirB genes.

qPCR

Protocol PCR Stages Temp (°C) Cycles Time Primer Sequence Amplicon

size

tlh Initial denaturation 95 1 7 min Forward

MV2B-TLF

5’-GTT GCA CTC

GGT GAC AGC TTG-3’

248 bp

Denaturation 95 50 5 s

Annealing 60 50 10 s Reverse

MV2B-TLR

5’- AGT TTT GCG TAG

GTT AAG TAC-3’

Extending 72 50 25 s

Final extending 72 1 7 min

pirA Initial denaturation 95 1 3 min Forward

VpPirA-F

5’-TTG GAC TGT

CGA ACC AAA CG-3’

135 bp

Denaturation 95 40 5 s Reverse

VpPirA-R

5’-GCA CCC CAT

TGG TAT TGA ATG3’

Annealing and extending 60 40 30 s

pirB Initial denaturation 95 1 3 min Forward

VpPirB-F

5´-ACT AGG CAA GGC

TCA TAA ATA TGA CG-3´

102 bp

Denaturation 95 40 5 s Reverse

VpPirB-R

5´-ATT GCT CAG GTC

CAT TGG CA ATA A-3´

Annealing and extending 60 40 30 s

Temp: temperature, bp: base pair.

6Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73: e20253577, enero-diciembre 2025 (Publicado Dic. 02, 2025)

statistical analyses were conducted using SPSS

Statistics version 25 (IBM Corp., 2017).

RESULTS

Molecular analysis in shrimp: Of the

shrimp groups studied, 44.4 % (16 / 36) were

positive for the presence of V. parahaemo-

litycus, in five of them the bacterium was

detected only in the hepatopancreas, in three

exclusively in the intestine, and in eight in both

intestine and hepatopancreas. Fig. 1 shows

the results of the frequencies of detection of

V. parahaemolitycus in individual samples of

juvenile shrimp organs and postlarvae groups.

Of the groups of juvenile shrimps ana-

lyzed, 55.5 % (20 / 36) were determined to be

positive for the pirA gene amplicon, of which

six groups presented the amplicon only in the

hepatopancreas, three only in the intestine, and

11 in both the intestine and hepatopancreas.

The results of the detection frequencies of

the pirA gene amplicon in individual samples of

juvenile shrimp organs and postlarvae groups

are shown in Fig. 2. Of the 31 samples positive

for this gene in juvenile shrimp organs, 30 were

also positive for the pirB gene amplicon and

18 were identified as V. parahaemolyticus, the

latter suggesting that other species of the genus

Vibrio present the pirA gene amplicon.

Of the groups of juvenile shrimps ana-

lyzed, 55.5 % (20 / 36) were determined to be

positive for the pirB gene amplicon, of which,

four showed the amplicon only in the hepato-

pancreas, three only in the intestine, and 13 in

both the intestine and hepatopancreas.

The results of the detection frequencies of

the pirB gene amplicon in individual samples of

juvenile shrimp organs and postlarvae groups

are represented in Fig. 3. Of the 33 pirB-

positive samples in juvenile shrimp organs, 30

were also positive for the pirA gene amplicon

and 19 were identified as V. parahaemolyticus,

the latter suggesting that other Vibrio species

have the pirB gene amplicon.

Molecular analysis in waters: The fre-

quencies of detection of V. parahaemolyticus

and the pirA and pirB gene amplicon in the

pond water samples are presented in Fig. 1,

Fig. 2 and Fig. 3. Of the five water samples

that were positive for the pirA gene amplicon,

Fig. 1. Presence of the tlh amplicon in individual organ samples from juvenile shrimp, postlarvae groups and pond water.

N: total number of samples analyzed.

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three were identified as V. parahaemolyticus

and all five were also positive for the pirB gene

amplicon. Of the six water samples that were

positive for the pirB gene amplicon, four were

identified as V. parahaemolyticus and five were

positive for the pirA gene amplicon.

Sequencing: Sequencing of three PCR

products from hepatopancreas (248 / 248 bp),

intestine (247 / 247 bp) and water (248 / 248

bp) samples amplified with the qPCR pro-

tocol for the tlh gene [GenBank PP591942,

PP591943 and PP591944, respectively]

Fig. 3. Presence of the pirB amplicon in individual organ samples from juvenile shrimp, postlarvae groups and pond water.

N: total number of samples analyzed.

Fig. 2. Presence of the pirA amplicon in individual organ samples from juvenile shrimp, postlarvae groups and pond water.

N: total number of samples analyzed.

8Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73: e20253577, enero-diciembre 2025 (Publicado Dic. 02, 2025)

confirmed the presence of V. parahaemolyticus

species and were found to be 100 % identical

to other sequences of this same species depos-

ited in GenBank. Of all the samples positive

for the pirA gene in conventional PCR, we

were able to sequence a product from a 322

/ 323 bp hepatopancreas sample, [PP795453],

this sequence was 99.7 % identical to other

sequences of the pirA gene of the genus Vibrio

deposited in GenBank. With the conventional

PCR protocol used for the pirB gene, positive

control was amplified, but no sample from the

present study.

Factors associated with the pirA, pirB

and tlh genes of positive shrimp in qPCR:

Five factors were found to be associated with

the presence of the pirA and pirB gene in

shrimp (p < 0.05): rainy season (pirA: OR =

3.64, 95 % CI (confidence interval) = 1.36-

9.75; pirB: OR = 2.8, 95 % CI = 1.07-7.30),

presence of pirA in water (pirA: OR = 4.32, 95

% CI = 1.59-11.74; pirB: OR = 3.45, 95 % CI

= 1.29-9.22), presence of pirB in water (pirA:

OR = 11.36, 95 % CI = 3.68-35.09; pirB: OR =

7.95, 95 % CI = 2.76-22.92), presence of tlh in

water (pirA: OR = 3.34, 95 % CI = 1.25-8.92;

pirB: OR = 3.45, 95 % CI = 1.29-9.21) and col-

lection time: 42-49 days after stocking (pirA:

OR = 4.20, 95 % CI = 1.23-14.36; pirB: OR =

3.4, 95 % CI = 1.03-11.26). Two factors were

found to be associated with the presence of V.

parahaemolyticus bacteria: rainy season (OR

= 15.40, 95 % CI = 3.97-59.70) and presence

of the tlh gene in water (OR = 9.00, 95 % CI =

2.90-27.90) (SMT1, SMT2, SMT3).

Physicochemical parameters of the

water in the ponds studied: No significant

differences were found in terms of the values

of physicochemical parameters between the

groups of positive and negative waters for the

amplicons of the pirA, pirB, and tlh genes.

Table 2 shows the physicochemical parameters

of the water in the ponds of the two farms

studied; the averages of each parameter were

calculated according to the season of the year.

Significant differences (p < 0.05) were only

found in the salinity parameter of pond water

between the dry and rainy seasons.

The qPCR protocols implemented in the

present investigation proved to be efficient

and economical and allowed the identification

of V. parahaemolyticus and the pathogenic

genes pirA and pirB in shrimp and pond water

samples, as well as in samples that had not been

analyzed (shrimp intestines) in Costa Rica. In

addition, factors associated with the presence of

V. parahaemolyticus bacteria (rainy season and

water positive for the tlh gene) and pathogenic

genes (rainy season, water positive for the tlh,

pirA and pirB genes and time of collection: 42

to 49 days after stocking) were identified.

DISCUSSION

The three protocols implemented present-

ed good efficiencies and specificity, which

agrees with what has been reported by other

authors (Han, Tang, Pantoja et al., 2015; Han,

Tang, Tran et al., 2015; Micky et al., 2014). It

is important to mention that the primers used

in the amplification protocol of the partial

sequence of the pirA gene were previously used

with TaqMan probes (Han, Tang, Pantoja et al.,

2015), however, in the present work a much

more economical technique, the SYBR Green

I, was used. Our study shows this technique

provides reliable results.

The presence of plasmidic genes pirA, pirB

and V. parahaemolyticus in shrimp samples and

pond waters is reporte high frequencies. It was

also possible to determine the genes in the

intestines of juvenile shrimp, an organ that had

not been studied for genes in the country so far.

In relation to the groups of shrimp ana-

lyzed, high frequencies of the pirA and pirB

genes (55.5 %) and V. parahemolyticus (44.4

%) were determined; these data are consistent

with those reported by other authors when

finding high prevalences of AHNPD in shrimp

(Morales-Covarrubias et al., 2019; Peña-

Navarro et al., 2020).

Sirikharin et al. (2015) point out that when

working with field samples, including shrimp

tissues such as hepatopancreas and stomach,

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73: e20253577, enero-diciembre 2025 (Publicado Dic. 02, 2025)

whole postlarvae and environmental samples

such as pond water in which the levels of

AHPND bacteria are low, a preliminary enrich-

ment step should be conducted. This is why all

the samples in this study were pre-enriched in

APW with NaCl. This culture medium is rec-

ommended as enrichment broth for Vibrio, so

perhaps this contributed to the high detection

of the genes of interest in addition to the fact

that the qPCR technique has a high detection

sensitivity when compared to conventional

PCR (Valasek & Repa, 2005).

The difference observed in the number of

cases identified as positive for the pirA gene

with respect to pirB could be explained by

one of the amplicon detection methods being

more sensitive than the other or by the pres-

ence of strains having only one of the genes

(Aranguren-Caro et al., 2020), however, further

studies are recommended to corroborate this.

With respect to the bacterial species iso-

lated from shrimp and water samples, not

all were identified as V. parahaemolyticus.

Although V. parahaemolyticus was initially

identified as the causative agent of AHPND in

shrimp, more recent studies have reported the

plasmid with the genes of interest in other Vib-

rio species, which caused AHPND in shrimp,

these species include Vibrio harveyi (Kondo

et al., 2015), Vibrio owensii (Liu et al., 2018),

Tab l e 2

Physicochemical parameters of the water quality of two ponds belonging to two shrimp farms (farm A and farm B) producing

L. vannamei shrimp in the Gulf of Nicoya, Costa Rica, during the year 2021.

Parameters Temperature (°C) pH Dissolved oxygen (mg/L) Salinity (ppt)

Optimal growth V. parahaemolyticus135-37 7.5-8.6 Facultative anaerobic 5-25

Optimal from shrimp farming228-30 7.5-8.5 5.0-10.0 15-25

Farm Sampling time Temperature (°C) pH Dissolved oxygen (mg/L) Salinity (ppt)

ASampling one 28.50 7.05 10.90 27.00

ASampling two 34.20 8.65 8.80 28.90

ASampling three 33.10 8.38 10.30 30.30

ASampling four 33.50 8.52 6.84 29.40

BSampling one 26.00 8.15 8.00 25.50

BSampling two 31.60 8.07 7.40 32.00

BSampling three 33.40 7.86 10.00 30.70

BSampling four 33.90 8.09 8.92 31.60

Average dry season 31.80 8.09 8.89 29.42

Max. 34.20 8.65 10.90 32.00

Min. 26.00 7.05 6.84 25.50

ASampling one 32.10 8.42 7.80 17.00

ASampling two 21.60 8.93 11.75 19.60

ASampling three 32.20 8.34 8.36 17.30

ASampling four 33.40 7.63 8.50 19.70

BSampling one 32.10 8.50 9.71 17.90

BSampling two 34.10 9.07 11.80 16.40

BSampling three 26.00 8.78 8.53 16.60

BSampling four 32.00 8.43 7.83 17.50

Average rainy season 30.44 8.51 9.28 17.75

Max. 34.10 9.07 11.80 19.70

Min. 21.60 7.63 7.80 16.40

Sampling one: day of stocking, sampling two: three to four weeks after stocking, sampling three: six to seven weeks after

stocking, and sampling four: nine to ten weeks after stocking. Values of parameters found to be outside the optimal range

for shrimp culture are highlighted in bold. / 1 Source taken from Urquhart etal. (2016) and Zamora-Pantoja etal. (2005). /

2 Source taken from Valverde (2020).

10 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73: e20253577, enero-diciembre 2025 (Publicado Dic. 02, 2025)

Vibrio campbellii (Dong et al., 2017) and

Vibrio punensis (Restrepo et al., 2018). Lee et

al. (2015) indicate that the pVA1 plasmid pos-

sessed by V. parahaemolyticus strains contain a

group of genes related to conjunctival transfer,

so it can potentially transfer not only between

V. parahaemolyticus strains but also to different

Vibrio species. Therefore, it is recommended in

future research to determine which species the

strains carrying the pirA and pirB genes that

are negative for V. parahaemolyticus belong to.

Thus, determining which other species in Costa

Rica present the pathogenic genes.

The detection of V. parahaemolyticus bac-

teria in a group of shrimp postlarvae is consis-

tent with reports by other authors that indicate

that infection of postlarvae with V. parahae-

molyticus bacteria is possible in the laboratory,

when they are in the last stage of metamor-

phosis (Aguirre-Guzmán et al., 2001). On the

contrary, the non-detection of the pirA and pirB

genes in the shrimp postlarvae indicates that

they were seeded free of pathogenicity genes,

which tends to think that the bacteria with

pathogenic genes could have been acquired

from water, food or sediments in the ponds and

not from the seed (Alonzo et al., 2017; Chonsin

et al., 2016; Thitamadee et al., 2016).

Alonzo et al. (2017) found in their study

that V. parahaemolyticus in shrimp rearing

water adhered to added feed, which facilitated

its entry into the shrimp gut; thereby, showing

that the oral cavity serves as an important por-

tal of entry for pathogens from the shrimp rear-

ing environment. In the farms studied, feed was

provided to the shrimp in troughs located at

different points in the ponds, at scheduled times

so that the shrimp in the water had access to it,

which could also provide a possible explana-

tion for the presence of this bacterium and the

genes of interest in the shrimp studied.

Another source of AHNPD infection could

also be sediments, as pathogenic V. parahae-

molyticus has been detected in shrimp pond

sediments (Thitamadee et al., 2016). It is for

this reason that it is recommended to always

implement proper preparation of the culture

area before stocking and properly manage the

pond during culture to exclude pathogenic V.

parahaemolyticus strains from the shrimp cul-

ture system. Because of the above, it is recom-

mended in future research to also analyze other

types of samples such as sediments or feed in

search, for the bacteria and pathogenic genes

(de Souza-Valente & Wan, 2021; Thitamadee

et al., 2016).

Zamora-Pantoja et al. (2005) indicate that

V. parahaemolyticus grows at temperatures

between 10 and 44 °C, but with an optimum

growth temperature of 35 °C and 37 °C.

Therefore, in this study, a higher probability

of finding qPCR-positive shrimp in summer,

when temperatures are higher, was expected.

In contrast, qPCR positive shrimp samples for

pirA, pirB and tlh were 4 (OR = 3.64), 3 (OR

= 2.8) and 15 (OR = 15.4) times more likely to

occur in the rainy season than qPCR negative

samples, respectively.

However, when comparing the water tem-

perature in the ponds during the dry and

rainy seasons, no significant differences were

observed; moreover, the water temperatures

recorded in both seasons were within the val-

ues considered optimal for the growth of V.

parahaemolyticus. Therefore, temperature in

this study was not a determining factor in the

probability of finding qPCR-positive shrimp

for the genes studied. The only factor analyzed

that was significantly different between the two

seasons was salinity, which could be related

to a higher probability of the presence of the

genes studied in the rainy season.

Studies have shown that in tropical zones,

salinity is an important factor that regulates

the dynamics of Vibrio and that a moderate

decrease in this parameter can promote the

abundance of Vibrio spp. On the other hand,

in some research works, a negative correla-

tion between salinity and V. parahaemolyticus

abundance has been observed (Machado &

Bordalo, 2016; Reyes-Velásquez et al., 2010).

In our study, it was determined that during

rainy season salinity presented on average a

significantly lower value (17.75 ppt) compared

to those from dry season (29.43 ppt), probably

due to the amount of rainfall that occurs during

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73: e20253577, enero-diciembre 2025 (Publicado Dic. 02, 2025)

rainy season in Costa Rica, which produces

an increase in the water levels of the shrimp

ponds and thus a decrease in the concentration

of salinity.

If the optimal environmental range of V.

parahaemolyticus is considered, it generally

increases in abundance with salinity between 5

and 25 ppt (Urquhart et al., 2016). According

to this, the average salinity that was presented

during rainy season was more favorable for the

growth of V. parahaemolyticus than that deter-

mined during dry season, which was presented

above the optimal range for this bacterium,

which could explain the higher probability that

the samples of shrimp positive for pirA, pirB

and V. parahaemolyticus were presented in the

rainy season than the negative ones.

Shrimp qPCR positive for pirA, pirB and

tlh were identified to be 3 (OR = 3.35), 3 (OR

= 3.45) and 9 (OR = 9.00) times more likely

to occur in tlh gene positive pond water than

qPCR negative for these genes, respectively.

On the other hand, qPCR positive shrimp for

pirA and pirB were identified to be 4 (OR =

4.32) and 3 (OR = 3.45) times more likely to

occur in pond water positive for the pirA gene

than qPCR negative shrimp, respectively. In

addition, they were 11 (OR = 11.36) and 8

(OR = 7.95) times more likely to occur in pond

water positive for the pirB gene than negative

qPCRs, respectively. V. parahaemolyticus is

known to be a ubiquitous bacterial pathogen

naturally found in marine environments (Bak-

er-Austin et al., 2010; World Organization for

Animal Health, 2023). Therefore, this patho-

genic bacterium could be expected to enter

through the seawater used to fill and replace

the water in the ponds. Under favorable envi-

ronmental conditions, the bacteria would have

proliferated, thus facilitating its detection.

Such results are in agreement with those

found in a previous study that reported an asso-

ciation of the presence of Vibrio spp bacteria

with high replacement rates and water volume

in the ponds (Peña-Navarro et al., 2020), which

could lead to an imbalance in the abundance of

the different bacterial communities and a pro-

liferation of pathogenic Vibrio. Accordingly,

it has been reported that AHNPD can cause a

dysbiosis in the microbiota of the hepatopan-

creas of shrimp Litopenaus vannamei (Ávila-

Villa et al., 2023).

On the other hand, in our study, the pirA

and pirB genes began to be detected in both

shrimp and pond water between sampling two

and three, which corresponds to the period

between 21 and 49 days after restocking the

ponds. Authors such as Ananda-Raja et al.

(2017), Han, Tang, Tran et al. (2015), and Zor-

riehzahra and Banaederakhshan (2015), note

that V. parahaemolyticus appears between 20

and 30 days after stocking ponds with post-

larvae. Thitamadee et al. (2016) point out that

mortalities occur between 35 and 45 days after

postlarvae stocking. These studies support our

results by presenting the first detection in the

second sampling (21 and 30 days). However,

it was not until the third sampling (42-49 days)

when an association with the presence of the

genes was observed.

It was not possible to find differences

between the averages or medians of the salinity,

temperature, dissolved oxygen and pH factors

between positive and negative detection of the

gene of interest in the water. This could be due

to the fact that the samples came from a single

point in each sampling. Therefore, it is recom-

mended that more pond water sampling, which

better represents the distribution of the data, be

conducted in future research.

With respect to the optimal values of the

pond water for shrimp culture, the averages

of two variables were found to be above the

optimal values during dry season: tempera-

ture and salinity. Higher temperature values

could generate stress conditions in shrimp and

higher salinity values could represent a higher

energy expenditure in osmoregulation and thus

a lower growth rate. These alterations can be

exploited by opportunistic pathogens such as V.

parahaemolyticus, increasing their population

and colonizing their hosts (Carbajal-Hernández

& Sánchez-Fernández, 2013; Lazur, 2007).

Therefore, adequate management of the physi-

cochemical parameters of the water in shrimp

culture ponds is recommended.

12 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73: e20253577, enero-diciembre 2025 (Publicado Dic. 02, 2025)

Our results showed that Vibrio parahaemo-

lyticus bacteria and the pathogenic genes pirA

and pirB were successfully detected in shrimp

and pond water samples collected during both

seasons of the year in Costa Rica. However,

detection was more frequent during the rainy

season, probably due to a significantly lower

concentration of dissolved salts in pond water

compared to the concentrations found during

the summer season. This suggests that salinity

could be an important environmental variable

to consider in the ecology of pathogenic Vibrio

parahaemolyticus bacteria and the incidence

of AHNPD. In addition, high frequencies of

the genes studied were found, so it is recom-

mended that animal health authorities in Costa

Rica pay special attention to AHNPD, which

has been declared notifiable by the World

Organization for Animal Health (WOAH).

Ethical statement: The authors declare

that they all agree with this publication and

made significant contributions; that there is no

conflict of interest of any kind; and that we fol-

lowed all pertinent ethical and legal procedures

and requirements. All financial sources are

fully and clearly stated in the acknowledgments

section. A signed document has been filed in

the journal archives.

See supplementary material

a70v73n1-suppl. 1

ACKNOWLEDGMENTS

This research was supported by the Fondo

Especial de Estímulo a la Investigación 2019

of the Vicerrectoría de Investigación of the

Universidad de Costa Rica. Thanks are due to

the shrimp producers who participated in the

research. Also, to Donald Arguedas Cortés for

suggesting the initiation of this project and for

his support with the endpoint PCR protocols

and to José Pablo Mora Quesada for providing

advice on the statistical analyses.

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