Aquaculture 579 (2024) 740221

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Aquaculture
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Identification of new Vibrio campbellii strains harboring the pVA1 plasmid

isolated from Penaeus vannamei postlarvae affected by outbreaks of acute
hepatopancreatic necrosis disease (AHPND) in Mexico
Sonia A. Soto-Rodriguez a, *, Bruno Gomez-Gil a, Rodolfo Lozano-Olvera a,
Karla G. Aguilar-Rendón a, Jean P. González-Gómez b
a
CIAD, AC Mazatlan Unit for Aquaculture and Environmental Management, Av. Sabalo-Cerritos 82112, Mazatlan, Mexico
b
CIAD, AC Culiacan Unit, Carretera a Eldorado Km 5.5, Campo El Diez, 80110 Culiacán, Sinaloa, Mexico

A R T I C L E I N F O A B S T R A C T

Keywords: At present, AHPND continues to produce large losses in Mexican shrimp farms, but no scientific report on
Shrimp postlarvae mortality in commercial hatcheries has been published. Here, we present the clinical signs, histopathological
pirAB genes lesions, bacteriological analyses and identification through whole-genome sequencing of the isolates obtained in
Vibrios
three natural outbreaks of AHPND in Penaeus vannamei postlarvae obtained from three local hatcheries. After
WGS
three to four days of acclimation, mortality was recorded and clinical signs such as an empty digestive tract,
Penaeus vannamei
whitish hepatopancreas, anorexia and lethargy were observed in shrimp postlarvae. Histopathological analysis
and detection of pirAB genes using the PCR AP4 method after sample enrichment were used as presumptive
diagnoses of AHPND. Significantly more Vibrio-like density (p = 0.045) and green colonies (p = 0.021) were
found in the hepatopancreas of postlarvae affected by AHPND compared to apparently healthy organisms in
outbreak 1 and 2, respectively. Finally, 29 isolates from the hepatopancreas and stomachs of shrimps with or
without AHPND clinical signs were sequenced, and two new Vibrio species were obtained, one closely related to
the Harveyi clade and one to the Orientalis clade. Vibrio campbellii were the most abundant species followed by
V. parahaemolyticus. The six genomes identified as V. campbellii harbour the pVA1-like plasmid carrying the pirAB
genes, responsible for producing the delta-endotoxin that causes AHPND. The pVA1-like plasmids were similar to
V. parahaemolyticus strains from the Americas. pVA1 plasmid hosts the Tn3 transposon like the V. campbellii
strain from Ecuador, but absent in the China strain, suggesting a horizontal plasmid transfer between coexisting
V. parahaemolyticus and V. campbellii within a geographic region. These findings provided the first report of
V. campbellii strains carrying a pVA1-like plasmid that causes AHPND in postlarvae from commercial hatcheries
in Mexico and may help in the adoption of active genomic surveillance of this disease.

1. Introduction
Diseases that affect postlarvae or early juvenile stages of farmed
shrimp are one of the main limitations for the sustainability of the
shrimp industry. Acute hepatopancreatic necrosis disease (AHPND),
caused mainly by Vibrio parahaemolyticus (Vp AHPND), continues to be
the bacterial disease of greatest economic importance that affects both
tiger shrimp (Penaeus monodon) and the Pacific white shrimp (Penaeus
vannamei) (FAO, 2013). The virulence of Vp AHPND is due to con­
jugative plasmids of approximately 70 kbp (pVA1) that express a binary
PirAB toxin that is homologous to the insecticidal Pir toxin (Han et al.,
2015a). The PirAB toxin, stable in seawater, affects the hepatopancreas,
the target organ of the disease, which is an essential digestive gland
involved in metabolism with multiple physiological and immunological
functions that be affected during infection (Muthukrishnan et al., 2019).
AHPND is characterised as causing acute necrosis of the hepato­
pancreas leading to its dysfunction (Joshi et al., 2014; Soto-Rodriguez
et al., 2015). In juvenile and adult shrimp, the clinical signs of the dis­
ease include lethargy, pale hepatopancreas, anorexia and an empty
digestive tract (Aguilar-Rendón et al., 2020). Histological observations
of shrimp affected by AHPND include massive sloughing and roundness
of the epithelial cell of the hepatopancreatic tubules into the lumen

* Corresponding author.
E-mail address: ssoto@ciad.mx (S.A. Soto-Rodriguez).

https://doi.org/10.1016/j.aquaculture.2023.740221
Received 21 January 2023; Received in revised form 11 October 2023; Accepted 12 October 2023
Available online 16 October 2023
0044-8486/© 2023 Elsevier B.V. All rights reserved.
S.A. Soto-Rodriguez et al.
(pathognomonic lesion), reduction of the reserve vacuoles and, as the
disease progresses, secondary bacterial colonisation of the damaged
hepatopancreas occurs. Confirmatory diagnosis of AHPND in shrimp
should also include, apart from the pathognomonic lesion observed in
the acute stage of the disease, the molecular detection of pirA and pirB
genes coupled with experimental infections (OIE, 2021). In Mexico, as in
other countries, a PCR laboratory test is usually performed before the
postlarvae are sent to a farm for cultivation. However, this procedure
cannot guarantee that the postlarvae sent are free from AHPND. PCR
protocols based on pirA and pirB genes are considered the most sensitive;
however, an enrichment step before extracting DNA is recommended for
environmental samples, such as sediments and biofilms, where bacteria
are present in small quantities (OIE, 2021).
During the shrimp culture, AHPND can reach 90 to 100% mortality;
however, shrimp are more susceptible in their early life stages to
intoxication by AHPND strains, with a threshold infective density of over
104 CFU mL− 1 (Soto-Rodriguez et al., 2015). Several AHPND-causing
Vibrio spp. have been identified from affected penaeid shrimp,
including Vibrio harveyi in Thailand (Kondo et al., 2015), Vibrio owensii
in China (Liu et al., 2015) and Vibrio punensis in South America (Restrepo
et al., 2018). Vibrio campbellii strains have been implicated in AHPND in
Guanxi, China (strain 20130629003S01; Dong et al., 2017a) and in a
Latin American country (strain LA16-V1; Ahn et al., 2017) isolated in
the Guayas province of Ecuador (https://www.ncbi.nlm.nih.gov/bios
ample/SAMN06909310/). Additionally, another four V. campbellii
strains have also been isolated in a “Latin American country” (Han et al.,
2017) and carry the pirAB genes as determined by a PCR amplification of
these genes; at least one causes AHPND in shrimp (the other were not
tested). The conjugative plasmid carrying the pirAB toxigenic genes has
also been demonstrated that it can be horizontally transferred between
the original V. campbellii strain to a receptor V. owensii strain under
controlled laboratory conditions (Dong et al., 2019a).
In Mexico, the disease was detected in 2013 in shrimp farms causing
70% production losses (Nunan et al., 2014). Currently, AHPND con­
tinues to produce large losses in shrimp culture, but no scientific report
on mortality in shrimp farms and hatcheries has been published. The
disease represents a special threat to the Penaeid shrimp culture due to
its diverse causal agents, complexity, and unknown pathogenesis, in
addition to the widespread nature of this disease. Recently, hatcheries
have suffered sporadic low survival rates of shrimp postlarvae, which
have been attributed to the presence of red tide in the coastal zone;
however, its etiology has not been confirmed.
Due to the aforementioned information, this work studied the causes
of mortality and morbidity in shrimp postlarvae with clinical signs of
AHPND from commercial hatcheries in Mexico, using whole genome
sequencing (WGS) methods.
2. Materials and methods
2.1. Husbandry conditions
On June 2, 8, and 30, 2022, shrimp postlarvae batches were trans­
ported from three local shrimp (Penaeus vannamei) commercial hatch­
eries to CIAD, AC Mazatlan Unit facilities, to be used in research
experiments. In the hatcheries, shrimp were held in brown-coloured
seawater due to the regular use of probiotics during the culture of the
larvae. Postlarvae were transported in a round 1000-L plastic container
filled with fresh seawater (35 g L− 1 salinity, pH 7.8–8.0 and 28–30 ◦ C
temperature), which were similar too the transport conditions used by
the hatcheries.
After arrival at the CIAD facilities, shrimp were immediately trans­
ferred to 400-L fibreglass round tanks and were held according to the
hatchery culture conditions (natural seawater at a salinity of 35 g L− 1,
pH of 7.8–8.1, temperature 30 ◦ C and photoperiod of 12 h light/12 h
dark). An open-flow seawater system was used for each batch, and
postlarvae were fed three times a day with commercial shrimp
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Aquaculture 579 (2024) 740221
postlarvae food. No probiotics were employed. Observations of external
signs of disease were carried out twice a day, such as swimming
behaviour, chromatophores, the colour of the hepatopancreas, an empty
digestive tract and larval activity. All animals were held following
accepted standards of humane animal care. No endangered or protected
species were used.
2.2. Sample collection and isolation of Vibrio–like bacteria
The health status of the postlarvae from each batch was checked on
the first day of acclimatisation. For the detection of WSSV, IHHNV and
TSV, the PCR protocols described in the OIE (2022) were followed. The
AP4 system in a nested PCR reaction (Dangtip et al., 2015) was used as a
presumptive diagnosis of clinically affected animals as recommend by
OIE (2021). During shrimp acclimation, a natural outbreak occurred in
each batch between 72 at 96 h after arrival at the facilities, and mortality
and clinical signs that resembled AHPND were observed (Tran et al.,
2013; Soto-Rodriguez et al., 2015). The same diagnostic tests were
applied in each batch. Once the first mortality occurred, wet mounts
were assembled from the hepatopancreas of postlarvae. In addition,
shrimp were weighed, and samples from the hepatopancreas and
stomach were aseptically dissected from postlarvae with clinical signs of
AHPND and organisms without signs. One part of each tissue was fixed
in Davidson solution for histology analysis; the second part was homo­
genised in 1.0 mL of sterile saline solution (NaCl 2.5%) and 10-fold
serially diluted in sterile saline solution. One hundred microliters of
the suspensions were inoculated on TCBS (Difco™) agar in triplicate to
enumerate the Vibrio–like bacteria. All plates were incubated at 30 ◦ C for
24 h, and the colony forming unit (CFU) per g was recorded. Yellow and
green colonies were re-streaked on TSA (Difco™) + 2.0% NaCl agar
(TSA+) to obtain pure cultures. Isolates were also inoculated on
CHROMagar™ Vibrio and incubated at 30 ◦ C for 48 h. Finally, isolates
were cryopreserved at − 80 ◦ C until used.
In the second outbreak, a preliminary enrichment step of samples
prior to DNA extraction was carried out in the postlarvae without clin­
ical signs of the disease (sub-clinical infections) recommended by the
OIE (2021). Ten individual samples obtained from the stomach and
hepatopancreas were homogenised in alkaline peptone water +2.5%
NaCl supplement and incubated 8–10 h at 30 ◦ C with shaking. Later,
samples were centrifuged at 8609 xg for 10 min, and the pellet was
tested for pirAB genes detection using the AP4 system. For the second
and third outbreaks, additional samples were taken for future meta­
genomics analysis.
2.3. Histopathological analysis
Hepatopancreatic tissue of postlarvae with clinical signs of AHPND
and without clinical signs was processed by routine histology (Bell and
Lightner, 1988). The paraffin-embedded tissue sections were cut at 5 μm
and stained with haematoxilin-eosin-floxin, according to the method­
ology of Lightner (1996). Histological slides were observed under a light
microscope (Olympus CX31).
2.4. Sequencing of the whole genome
A total of 29 pure bacterial isolates were obtained. DNA was
extracted with the Promega Wizard Genomic extraction kit according to
the manufacturer's instructions. The extracted DNA was tagmented with
the Illumina Nextera XT kit, indexed and sequenced in the Illumina
Miniseq platform to obtain a low sequencing depth to identify the
isolates.
The resulting sequences were cleaned with a proprietary script
nextera_cleaner (https://github.com/GenomicaMicrob/nextera_cleaner
) and assembled with A5-miseq (Coil et al., 2015). The quality of the
assembled genomes was assessed with Checkm2 (Chklovski et al., 2022)
and then classified with GTDB-Tk v2.1.0 (Chaumeil et al., 2022), and
S.A. Soto-Rodriguez et al.
also some Average Nucleotide Identity (ANI) values were calculated
with OAT (Lee et al., 2016), comparing the type strains of each species.
In total, two V. campbellii AHPND+ genomes are already available,
and six new ones are included in this study. We compared the pVA1
plasmid architecture of these eight genomes including a
V. parahaemolyticus genome (M0904, González-Gómez et al., 2020) as a
reference. A pangenome of V. campbellii was obtained with anvi'o v7.1
(Eren et al., 2015) that, apart from the eight AHPND+ genomes,
included the type strain of the species (ATCC 25920T).
2.5. Statistical analyses
For the enumeration of the Vibrio–like bacteria and green colonies,
data were first analysed to determine whether they were normally
distributed, using the Shapiro-Wilk normality test, and homogeneity of
variances, using a Levene's test. If normality was rejected, Mann-
Whitney rank sum test post-hoc pairwise comparisons were used to
compare the medians between groups. All analyses were performed
using a significance of 0.05.
3. Results
In the first, second, and third outbreak, the weight of the postlarvae
was from 80 to 190 mg, 180 to 310 mg, and 80 to 240 mg, respectively.
At the beginning of acclimation, no clinical signs of diseases were
observed, and detection by PCR of WSSV, IHHNV, TSV and AHPND were
negative in all the batches. However, when the enrichment method was
applied (second batch), seven out of ten samples of postlarvae without
clinical signs were presumptively positive for AHPND.
During the initial days of acclimation, the postlarvae exhibited
normal activity across all batches. However, after three or four days,
mortality occurred ranging from 70 to 80% in all tanks (Fig. 1a). Clinical
signs of AHPND were observed, such as empty digestive tract (stomach
and intestine), pale and atrophied hepatopancreas, erratic swimming,
anorexia and lethargy (Fig. 1b).
3.1. Wet mount of hepatopancreas
Wet mounts of postlarvae with clinical signs of AHPND showed
lower light refringence of the hepatopancreatic tissue than postlarvae
without clinical signs (Fig. 2). Diseased shrimp showed pale-to-whitish
colour and few vacuoles in the cells of the hepatopancreatic tubules. A
flat tubular epithelium, absence of lipid droplets and secretory vacuoles
Fig. 1. Photographs of P. vannamei postlarvae from an AHPND natural outbreak. a. Shrimp from the first outbreak, showing dead (arrow), moribund and normal
shrimp. b. Box from Fig. 1a, diseased shrimp with clinical signs of AHPND (arrows) and shrimp without clinical signs exhibited full digestive tract (stomach and
intestine) and normal hepatopancreas with the typical pigmentation (arrowhead).
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Aquaculture 579 (2024) 740221
in R and B cells (respectively), massive sloughing of epithelial cells and a
deformed tubule (due to accumulation of necrotic cells in the tubular
lumen) were clearly observed, suggesting an acute stage of AHPND
(Fig. 2a). Shrimp with a whitish hepatopancreas showed the lowest
refringence and melanised necrotic lesions in the tubules (Fig. 2b-c).
Shrimp without clinical signs of AHPND showed more refringence in the
hepatopancreatic tissue and abundant lipid droplets and secretory vac­
uoles (Fig. 2d).
3.2. Histopathological observations
All postlarvae with clinical signs of AHPND from the three outbreaks
showed histopathological changes associated with the acute, terminal,
or remission stage of AHPND (AHPND+) (Fig. 3). The hepatopancreas
lesions in the acute stage showed massive sloughing cells into the tubule
lumen, mild hemocytic infiltration and lack of vacuoles in the tubular
epithelial cells (Fig. 3a – b). The hepatopancreas with terminal stage
lesions showed severe melanisation and necrosis of hepatopancreatic
tissue with an abundant mass of bacteria in the necrotic tissue (Fig. 3c).
Postlarvae without clinical signs of AHPND did not display pathological
changes (AHPND-) (Fig. 3d).
3.3. Enumeration of the Vibrio–like bacteria
The Vibrio-like density and green colonies on TCBS showed different
results for each AHPND outbreaks. As the data lacked normality, the
Mann-Whitney rank sum test was used to compare Log CFU g− 1 between
the hepatopancreas and stomach, as well as the percentage of green
colonies on TCBS of AHPND+ and AHPND- postlarvae for the three
outbreaks (Table 1). Outbreak 1 showed significant differences (p =
0.045) between Vibrio-like density of the hepatopancreas and stomach of
AHPND+ organisms. In the three outbreaks, the Log CFU g− 1 values of
the hepatopancreas were higher than the stomach of AHPND+ shrimp.
In addition, the hepatopancreas of AHPND+ postlarvae had higher
values than the hepatopancreas of AHPND- organisms. Outbreak 2
showed significant differences (p = 0.021) of green colonies between the
hepatopancreas and stomach of AHPND+ organisms. Green colonies in
the hepatopancreas of AHPND+ postlarvae ranged from 58 to 77%,
compared to AHPND- postlarvae, which ranged from 13 to 51%.
3.4. Species identification of the outbreaks
A total of 11 known species were identified (Table 2), and two
S.A. Soto-Rodriguez et al. Aquaculture 579 (2024) 740221

Fig. 2. Wet mounts of the hepatopancreas of diseased P. vannamei postlarvae from the first and second outbreaks with clinical signs of AHPND (a – c). d. Organisms
without clinical signs. a. Microphotography of the hepatopancreatic tubules showing massive sloughing and accumulation of the epithelial cells into the lumen
(arrow). b – c. Lack of vacuoles in the R and B cells (arrow), deformities in the tubules (arrowhead), and melanisation in the tubules (asterisk). d. Tubules with
abundant vacuoles in epithelial cells and normal morphology.

Fig. 3. Microphotography of the hepatopancreas of P. vannamei postlarvae from natural outbreaks. a–b. Hepatopancreas in the acute stage of AHPND, massive
sloughing of the epithelial cells, low hemocytic infiltration and accumulation of cells into the tubular lumen were observed. c. Hepatopancreas with severe necrosis,
melanisation, hemocytic infiltration and bacterial proliferation associated with the terminal stage of AHPND. d. Hepatopancreas without pathological changes,
normal tubular epithelium with abundant vacuoles in the R and B cells. Hematoxylin-eosin-floxin stain.

potential new species of Vibrio were also obtained; of the 29 genomes, 27 Vibrio owensii of the Harveyi clade, and Vibrio sp. nov. 2 (one genome) to
belonged to Vibrio and two to Photobacterium damselae. The potentially Vibrio tubiashii, belonging to the Orientalis clade.
new species had an ANI value below the standard threshold of 95% to The six Mexican Vibrio campbellii strains are very closely related (>
delimit a species; Vibrio sp. nov. 1 (one genome) is closely related to 0.9996 ANI); even so, each have some genes, between 4 and 20, found

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S.A. Soto-Rodriguez et al. Aquaculture 579 (2024) 740221

Table 1 transposon grew from 5537 nucleotides to 6600. The second transposon
Comparison between medians of Log CFU g− 1 and percentage of green colonies (Tn3) found on this same plasmid is absent in the Chinese strain
on TCBS from the hepatopancreas and stomach of Penaeus vannamei postlarvae 20130629003S01 (Fig. 5).
for each outbreak. Median ± standard error of the median, n was between five The pirAB genes, responsible for producing the delta-endotoxin that
and ten samples. causes AHPND, were only detected in all the six genomes identified as
Outbreak Vibrio campbellii in what appears to be a conjugative plasmid very similar
1 2 3 to the one previously described for Vibrio parahaemolyticus strain M0904
collected in 2013 in a nearby locality.
Log CFU g¡1
V. campbellii isolates were the most abundant species followed by
Hepatopancreas 7.00 ± 0.36 6.59 ± 0.57 4.39 ± 0.81 V. parahaemolyticus with five isolates (see Fig. S1 in Suppl. Material).
AHPND+ Stomach 6.30 ± 0.50 5.13 ± 0.60 3.66 ± 0.35
p 0.045 0.141 0.348
Colony colour on CHROMagar™ Vibrio was diverse; most of the isolates
Hepatopancreas 2.62 ± 0.38 4.04 ± 0.30 2.12 ± 0.28 showed mauve colour (including P. damselae), except Vibrio fortis and
AHPND- Stomach 3.58 ± 0.36 5.40 ± 0.44 3.02 ± 0.30 Vibrio rotiferianus, which displayed turquoise colour, Vibrio sp.nov. 2
p 0.087 0.057 0.079 was translucent, Vibrio parahaemolyticus was pale or navy blue and Vibrio
alginolyticus was cream.
% Green colonies
Hepatopancreas 57.87 ± 11.58 63.74 ± 20.59 77.45 ± 13.25
AHPND+ Stomach 80.93 ± 9.00 13.88 ± 8.95 76.91 ± 10.13
4. Discussion
p 0.088 0.021 0.759
Hepatopancreas 47.33 ± 7.56 13.14 ± 7.49 51.09 ± 13.84 The occurrence of outbreaks in farmed shrimp culture is complex;
AHPND- Stomach 59.90 ± 9.20 12.55 ± 6.29 46.65 ± 7.57 these are associated with multiple environmental factors, the host and
p 0.393 0.757 0.763
the presence of pathogens. The rapid growth of shrimp production has
AHPND+: postlarvae with AHPND; AHPND-: postlarvae without AHPND. promoted the development of the seed industry, and the annual output
of shrimp larvae has exceeded 1.5 trillion (Wang et al., 2021). Unfor­
only in that strain (Fig. 4; anvi'o), so they cannot be considered clones. tunately, the shrimp hatcheries are currently threatened by frequent and
serious diseases. Tthe risk of developing AHPND increases with the
culture condition of high water temperature, salinity and stocking
3.5. Plasmid analysis density of postlarvae (Boonyawiwat et al., 2017). Northwestern Mexico
dominates semi-intensive farmed shrimp production, with the majority
The pVA1 plasmid architecture of the analysed genomes did not of commercial hatcheries located in the region. Specifically, the state of
differ by more than a few nucleotides. The Tn6264 transposon carrying Sinaloa hosts around 900 shrimp farms and 50 shrimp nurseries, all
the pirAB operon is also identical between all the genomes, except that strategically located within biosafety facilities with the objective of
of strain 20130629003S01 of China, where a third IS5 family trans­ enhancing production. However, in 2020 and 2021, 38% and 31% of
posase was found inserted and disrupting the putative cell wall surface farms, respectively, were affected by infectious diseases (https://c
anchor family protein, located in the operon upstream of the pirAB esasin.mx/programacrustaceos/ reviewed December 2022). AHPND
(Figs. 5, 6). This double insertion also caused that part of the first operon remains a significant concern for the Mexican government and shrimp
and all the pirAB operon to change direction (Fig. 6), and also the farming industry (SENASICA, 2020). Although shrimp hatchery

Table 2
Whole genome sequencing of the isolates from Penaeus vannamei postlarvae affected by AHPND in Mexico.
Isolate Out. AHPND clinical signs Organ TCBS CHROMagar™ Vibrio AHPND plasmid Identification ANI % Accession number

M250101 1 + Hp, St Green Mauve YES Vibrio campbellii 96.20 JATABR000000000

M250103 1 + Hp, St Green Turquoise NO Vibrio fortis 96.90 JATABQ000000000
M250105 1 + Hp, St Yellow Turquoise NO Vibrio fortis 96.74 JATABP000000000
M250108 1 + Hp Yellow Mauve NO Vibrio owensii 96.58 JATABO000000000
M250109 1 + Hp Green Mauve YES Vibrio campbellii 96.15 JATABN000000000
M250114 1 + St Green Mauve YES Vibrio campbellii 96.16 JATABM000000000
M250116 1 + St Yellow Mauve NO Vibrio harveyi 98.73 JATABL000000000
M250201 1 − St Yellow Mauve NO Vibrio owensii 96.65 JATABK000000000
M250219 1 − St Yellow Mauve NO Vibrio mytili 98.91 JATABJ000000000
M250220 1 − Hp Green Mauve NO Vibrio brasiliensis 98.05 JATABI000000000
M260202 2 + Hp Green Mauve NO Vibrio parahaemolyticus 98.43 JATABE000000000
M260206 2 + Hp Yellow Mauve NO Vibrio owensii 96.57 JATABB000000000
M260204 2 + Hp Green Mauve NO Vibrio parahaemolyticus 98.44 JATABD000000000
M260205 2 + Hp Green Mauve NO Vibrio parahaemolyticus 98.41 JATABC000000000
M260112 2 − Hp Green Mauve NO Vibrio brasiliensis 98.10 JATABH000000000
M260118 2 − St Yellow Translucent NO Vibrio sp.nov. 2 94.17 JATABG000000000
M260121 2 − St Yellow Mauve NO Vibrio sp.nov.1 94.40 JATABF000000000
M260125 2 − St Yellow Turquoise NO Vibrio rotiferianus 96.93 JAVHXL000000000
M270202 3 + St Green Mauve NO Photobacterium damselae 97.99 JATAAZ000000000
M270203 3 + Hp Yellow Pale blue NO Vibrio parahaemolyticus 98.82 Pending
M270204 3 + Hp Green Mauve YES Vibrio campbellii 96.17 JAVHXJ000000000
M270206 3 + St Yellow Cream NO Vibrio alginolyticus 98.55 JAVHXI000000000
M270210 3 + St Green Mauve YES Vibrio campbellii 96.04 JAVHXH000000000
M270315 3 − St Yellow Mauve NO Vibrio sinaloensis 97.31 JAVHXG000000000
M270316 3 − St Yellow Mauve NO Vibrio sinaloensis 97.31 JAVHXF000000000
M270101 3 − St Yellow Mauve NO Vibrio owensii 96.09 JATABA000000000
M270102 3 − St Yellow Mauve NO Photobacterium damselae 98.37 JAVHXK000000000
M27032001 3 − Hp, St Green Navy blue NO Vibrio parahaemolyticus 98.29 JATAAY000000000
M27032002 3 − Hp, St Green Mauve YES Vibrio campbellii. 96.12 JATAAX000000000

Out.: outbreak; Hp: hepatopancreas; St: stomach; TCBS: Thiosulfate citrate bile salts sucrose agar; ANI: Average nucleotide identity.

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S.A. Soto-Rodriguez et al. Aquaculture 579 (2024) 740221

Fig. 4. Pangenome representation of Vibrio campbellii strains causing AHPND. Bins (external circle): Core genome = gene clusters present in all genomes analysed;
AHPND+ = genes clusters present in all AHPND+ strains; MX = gene clusters found only in the Mexican strains; strain codes = gene clusters found in only that strain.
Red squares = ANI values (> 96%). Genomes phylogenetically arranged based on the ANI values (dendrogram). Pangenome calculated with anvi'o v7.1 using the
type strain ATCC 25920T as reference. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

mortality has primarily been attributed to toxins from algal blooms in
recent years, the results presented here demonstrate that outbreaks in
postlarvae were associated with the presence of pirAB+ isolates, even at
densities below the infectious threshold (Soto-Rodriguez et al., 2015).
In the present study, outbreaks of AHPND occurred from 72 to 96 h
after postlarvae transportation. Clinical signs and histopathological le­
sions resembled those reported previously for AHPND (Tran et al., 2013;
Soto-Rodriguez et al., 2022; Lozano-Olvera and Abad-Rosales, 2023).
Common indicators included empty digestive tracts, pale to whitish
hepatopancreas and massive sloughing of epithelial cells, characteristic
of the acute stage of AHPND. Additionally, hepatopancreas with
melanised necrosis was observed during the terminal and remission
stages. The V. campbellii pirAB+ strains will be used in challenges with
postlarvae and juvenile shrimp to register the infectious process of
AHPND in future studies. Although OIE (2021) does not consider his­
topathology to be a confirmatory diagnosis of AHPND, this analysis
shows evidence of the clinical disease, including the degree and devel­
opment of lesions in the target tissue. Therefore, this method could be
used for detection and confirmation of clinical infections, based on the
presence of hepatopancreatic lesions typical of the acute phase of the
disease, such as massive sloughing of the epithelial cells and accumu­
lation of dead cells into the lumen, which have only been observed in
shrimp affected by AHPND (Tran et al., 2013; Joshi et al., 2014; Soto-
Rodriguez et al., 2015; Hong et al., 2016; Restrepo et al., 2018; Aguilar-

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Fig. 5. Syntheny of the pVA1 plasmid among the genomes analysed.

Fig. 6. Tn6264 transposon architecture of the pVA1 plasmid carrying the pirAB genes. Top, original transposon architecture present in V. parahaemolyticus strain
M0904 and in all V. campbellii strains, except strain 20130629003S01 of Guanxi, China (=CP020078, bottom). White arrows, transposase (IS903) genes; dark grey
arrows, hypothetical protein gene (hp); black arrows, genes with known function; small light grey arrows, potential promoters; small black arrows, repeat sequences;
textured boxes, truncated gene. CSAP, putative cell wall surface anchor family protein.

Rendón et al., 2020; Han et al., 2020).
The wet mount and histopathology analysis of postlarvae displaying
AHPND signs revealed varying disease progression within each
outbreak, suggesting a gradual infection that did not affect the entire
population. These events may have been the result of microbiome dys­
biosis within the tank ecosystem, potentially caused by the removal of
probiotics applied in the hatchery upon arrival; clear water could favour
fast-growing bacteria, including Vibrio sp. pirAB+ (De Schryver et al.,
2014). In this study, beneficial microorganisms from the rearing water
were cleared out under the transported and laboratory conditions (5–10
μm- filter seawater, 29 ± 1 ◦ C, aeration and 12 h light/12 h dark
photoperiod). Currently, in Mexico, a common shrimp hatcheries and
nurseries practise is the use of matured water systems; it means the
application of diverse commercial and homemade probiotics, as well as
postbiotics, enzymes, yeast, molasses and sometimes in combination
with bio floc technology. The water-rearing system comprises a complex
consortium of bacteria, yeast and protozoa cohabiting with the shrimp
larvae. Silva et al. (2012) reported that a commercial probiotic did not
influence the survival and growth of P. vannamei postlarvae, although
there was a reduction in the Vibrio-like count from the water and shrimp.
Meanwhile, Wang et al. (2020) found high Vibrionaceae proportion in
zoea1 and postlarvae 1 after five commercial probiotics. Shrimp
hatcheries and nurseries should take care in the use of commercial
products during the larval culture.
In this study, the results of the vibrios in postlarvae were dependent
on the hatchery; however, more Vibrio-like density and green colonies
were found in the hepatopancreas of AHPND+ postlarvae compared to
AHPND– shrimp, which may indicate that the secondary infection is
faster in postlarvae.
Current shrimp hatchery, nursery and farming practises promoted
such genetic exchanges, which highlighted a risk of the emergence of
new virulent populations of Vibrio sp., causing AHPND as reported here,
with potentially devastating consequences for shrimp culture (Fu et al.,
2020). Recently, Intriago et al. (2023) reported Vibrio-like species car­
rying Vp pirAB genes in P. vannamei postlarvae from Latin American
hatcheries. Unfortunately, these isolates were only biochemically iden­
tified using API20E. WGS should be used as the only method to reach
species-level precision (Culot et al., 2021), is the recommended method

7
S.A. Soto-Rodriguez et al.
for accurate identification and is used in few works to confirm vibrio
species (Gomez-Gil et al., 2014; Gomez-Jimenez et al., 2014; Restrepo
et al., 2016; González-Gómez et al., 2020). This is the first report, using
WGS, of V. campbellii strains, carrying the toxic pirA and pirB genes
isolated from shrimp postlarvae in Mexican commercial hatcheries;
previously, Vibrio species causing AHPND outbreaks occurred only in
shrimp farms (Nunan et al., 2014; Soto-Rodriguez et al., 2015; López-
León et al., 2016). Reliable identification of Vibrio spp. strains found in
aquaculture farms is very important since such identification could be
used to monitor the spread and occurrence of AHPND; new Vibrio species
pirAB+ will surely continue to appear in shrimp farms around the world.
In addition, four V. owensii and five V. parahaemolyticus isolates,
without the pirAB genes, were identified as part of the postlarvae bac­
terial community, even though those genes have been identified in other
strains (Liu et al., 2015; Restrepo et al., 2016). Other Vibrio species,
commonly associated with seawater, were also identified less
frequently, as part of the microbiota of the postlarvae from the AHPND
outbreaks. Again, Vibrio-like colonies causing AHPND displayed diverse
colours on CHROMagar™ Vibrio as reported by Soto-Rodriguez et al.
(2019). Most of the isolates were mauve colonies even though they grew
as yellow colonies on TCBS such as V. owensii, V. harveyi, Vibrio mytili
and Vibrio sinaloensis, including P. damselae. For detection or enumera­
tion of potential pathogenic bacteria in surveillance programmes, we do
not recommend the use of colour phenotype criteria.
Since the emergence of AHPND, epidemiological studies have pri­
marily centred on V. parahaemolyticus AHPND+ strains, given their
extensive reporting and the availability of a significant number of ge­
nomes. However, V. campbellii strains have demonstrated comparable
mortality rates to V. parahaemolyticus strains, warranting careful
consideration (Dong et al., 2017b). This investigation unveiled six newly
sequenced V. campbellii genomes, forming a distinct clade separate from
the previously documented Ecuadorian and Chinese strains carrying the
pirAB operon. Although the reported genome count for this species has
increased, it remains limited for conclusive evolutionary analysis.
However, this limitation is somewhat mitigated by the significant
impact of horizontal plasmid transfer, extensively documented in the
literature (Dong et al., 2019a; Muthukrishnan et al., 2019; Fu et al.,
2020). Consequently, plasmid analyses hold the potential to yield
invaluable insights into the disease dynamics. To date, outbreaks of
AHPND occur worldwide and can be caused by multiple Vibrio species.
Therefore, the importance of establishing health surveillance pro­
grammes in shrimp hatcheries to detect the presence of the virulence
plasmid should be emphasised. Curiously, OIE (2021) does not consider
WGS as a confirmatory diagnosis of AHPND although this method
identifies the presence of genes pirAB without any error. This method
will be included as a confirmative diagnosis of AHPND in shrimp.
Over the years, the architecture of pVA1 plasmids has been noted for
its high conservation. However, punctual differences have also emerged,
notably the presence or absence of the Tn3 transposon, inclusion of
IS903 transposases in Tn6264 and deletions in the pirAB region (Han
et al., 2015b; Han et al., 2017; Yan et al., 2019; González-Gómez et al.,
2020). The Tn3 transposon has been identified as a distinguishing factor
between V. parahaemolyticus AHPND+ strains in the Americas and those
in Asia, though its presence has also been reported in a Vietnamese
strain's plasmid (Kumar et al., 2018). This observation appears to extend
to V. campbellii strain LA16-V1 (Ecuador) hosts the transposon; whereas,
it is absent in 20130629003S1 (China), despite notable ANI values. This
phenomenon is elucidated by horizontal plasmid transfer between
coexisting species within geographic regions (Dong et al., 2019b). In the
Chinese V. campbellii strain, the IS903 transposase insertion was noted,
along with a reorientation of the pirAB operon; intriguingly, this alter­
ation did not impede its AHPND-causing capability (Dong et al., 2017a).
Comparable alterations have been detected in V. parahaemolyticus,
exemplified by the R14 strain where transposase insertion occurs up­
stream of the pirA gene, resulting in the absence of AHPND clinical signs
in P. vannamei (Kanrar and Dhar, 2018). Remarkably, deletions in the
8
Aquaculture 579 (2024) 740221
pirAB region were absent in V. campbellii plasmids, a contrast to obser­
vations in V. parahaemolyticus strains' plasmids reported by multiple
authors (Han et al., 2017; Yan et al., 2019), including instances of in
vitro passaging (Xiao et al., 2017).
Although the AP4 system is recommend as the PCR method for
AHPND by OIE (2021), in this study, postlarvae upon arrival from the
hatcheries were negative using that method. However, the preliminary
enrichment broth culture allowed the multiplication of pirAB+
V. campbellii, increasing the test's sensitivity. We identified that without
the enrichment culture there is a high probability of false negative re­
sults. Therefore, this protocol must be included in the detection of pirAB
genes by PCR. Commercial kits and official diagnoses for AHPND must
be revised to check the reliability of the PCR methods.
5. Conclusions
Apparent healthy P. vannamei postlarvae from shrimp hatcheries
developed clinical signs and histopathological lesions characteristic of
AHPND after 72 to 96 h of transportation.
The pVA1-like plasmid was identified in all V. campbellii genomes of
the strains, which confirmed the presence of pirAB genes for the first
time in postlarvae from Mexican commercial hatcheries, which is a great
threat to the shrimp industry. Most of the isolates of the outbreaks were
vibrios (93%) including two new Vibrio species as part of the microbiota
associated with postlarvae.
Genomes of V. campbellii strains were different from Ecuadorian and
Chinese strains and host the Tn3 transposon in the pVA1 plasmid like
V. campbellii strain LA16-V1 (Ecuador) and V. parahaemolyticus
AHPND+ strains from Americas; whereas, it is absent in
20130629003S1 (China). This suggests that pirAB genes were acquired
by horizontal plasmid transfer between coexisting V. parahaemolyticus
and V. campbellii within a geographic region. Finally, caution must be
observed when using PCR methods to detect pirAB genes for diagnosis of
AHPND in shrimp postlarvae; we strongly recommend a previous incu­
bation period of the digestive tract or the whole larval tissue to avoid
negative results.
Declaration of Competing Interest
The authors declare that they have no competing interests.
Data availability
No data was used for the research described in the article.
Acknowledgments
We thank to shrimp hatcheries for the postlarvae supply; Juan
Manuel Serrano, Carmen Bolán-Mejía for technical help in bacteriology
and Julissa Enciso-Ibarra for sequencing.
Ethics approval and consent to participate
This work did not involve the direct study of humans. All applicable
international, national, and/or institutional guidelines for the care and
use of animals were followed and all.
Funding
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
CRediT authorship contribution statement
Sonia Soto-Rodriguez: Conceptualization, Supervision, Writing-
Original draft preparation. Bruno Gomez-Gil: Formal analysis, Data
S.A. Soto-Rodriguez et al. Aquaculture 579 (2024) 740221

curation. Rodolfo Lozano-Olvera: Methodology, Investigation, FAO, 2013. Report of the FAO/MARD Technical Workshop on Early Mortality Syndrome
(EMS) or Acute Hepatopancreatic Necrosis Syndrome (AHPNS) of Cultured Shrimp
Writing-Original draft. Karla G. Aguilar-Rendón: Methodology,
(under TCP/VIE/3304), Hanoi, Vietnam, 25 to 27 June 2013; Report No. 1053; FAO
Investigation. Jean P. González-Gómez: Data curation, Methodology, Fisheries and Aquaculture, Rome, Italy.
Writing - review & editing. Fu, S., Wei, D., Yang, Q., Xie, G., Pang, B., Wang, Y., Lan, R., Wang, Q., Dong, X.,
Zhang, X., Huang, J., Feng, J., Liu, Y., 2020. Horizontal plasmid transfer promotes
the dissemination of Asian acute hepatopancreatic necrosis disease and provides a
Availability of data and materials novel mechanism for genetic exchange and environmental adaptation. mSystems. 5
(2) https://doi.org/10.1128/mSystems.00799-19 e00799–19.
Gomez-Gil, B., Soto-Rodríguez, S., Lozano, R., Betancourt-Lozano, M., 2014. Draft
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genomeA.00055-14 e00055–14.
Gomez-Jimenez, S., Noriega-Orozco, L., Sotelo-Mundo, R.R., Cantu-Robles, V.A., Cobian-
Consent for publication Guemes, A.G., Cota-Verdugo, R.G., Gamez-Alejo, L.A., Pozo-Yauner, L., Guevara-
Hernandez, E., Garcia-Orozco, K.D., Lopez-Zavala, A.A., Ochoa-Leyva, A., 2014.
The authors confirm that the work described has not been published High-quality draft genomes of two Vibrio parahaemolyticus strains aid in
understanding acute hepatopancreatic necrosis disease of cultured shrimps in
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