Journal of Invertebrate Pathology 174 (2020) 107424

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Journal of Invertebrate Pathology

journal homepage: www.elsevier.com/locate/jip

Acute hepatopancreatic necrosis disease (VPAHPND), a chronic disease in T

shrimp (Penaeus vannamei) population raised in latin America
Luis Fernando Aranguren Caro⁎, Hung N. Mai, Brenda Noble, Arun K. Dhar
School of Animal and Comparative Biomedical Sciences, University of Arizona, 1041 E. Lowell Street, Tucson, AZ 85721, USA

ARTICLE INFO ABSTRACT

Keywords: In Latin American shrimp farming, acute hepatopancreatic necrosis disease (AHPND) does not cause the acute
Penaeus vannamei mortalities observed in SE Asia. Herein we report for the first time a new phase of infection of AHPND, a chronic
AHPND phase based on two experimental AHPND-challenge trials using shrimp lines from Latin America. Three shrimp
Histopathology lines of Penaeus vannamei were challenged with a highly pathogenic strain of Vibrio parahaemolyticus causing
Genetic selection
AHPND (VPAHPND). PCR and histopathology assays were used for confirmation of AHPND in the trials. The first
Vibrio parahaemolyticus
study was to compare survival between the lines. A follow-up trial was conducted to document hepatopancreas
heterotrophic bacterial count and to measure the expression of VPAHPND binary toxin genes (pirAB genes) at
24 h.p.i. One of the Latin American shrimp lines, APE1, had significantly higher survival than recorded for the
other two lines (APE2 & APE3) and the specific-pathogen-free positive control line. Histopathology showed
typical AHPND acute and terminal phase lesions in VPAHPND challenged groups, although destructive cellular
changes were more pronounced in the SPF line. Histopathology of animals surviving AHPND revealed a unique
chronic phase of infection that resembles septic hepatopancreatic necrosis (SHPN), recognized as diagnostic of
digestive tract vibriosis. Data to support our finding, including a quantitative RT-PCR assay, confirmed the
expression of pirAB genes and the differential hepatopancreas heterotrophic plate count (HPC) among the dif-
ferent lines challenged. The results explain in part why the shrimp industry in some Latin American countries
continues to grow despite the presence of AHPND. In addition, the biology and pathology of AHPND resistant/
tolerant shrimp appear to be quite unique in this Latin American shrimp population.

1. Introduction
Since 2009, acute hepatopancreatic necrosis disease (AHPND) has
progressively spread as an epidemic, devastating shrimp production
across much of the shrimp farming region in Asia. Eventually, the dis-
ease reached the Western Hemisphere (Cuéllar-Anjel et al., 2012;
Nunan et al., 2014; Soto-Rodriguez et al., 2015), including an outbreak
identified on a farm in the USA (Dhar et al., 2019).
Previous estimates of the cumulative economic impact of AHPND
have ranged from ~4 billion USD for the Americas to ~8 billion for
Asia (Lightner, personal communication). However, in recent years,
shrimp production has improved in countries previously hard hit by
AHPND (Anon, 2018), as farmers have tested, improved and adapted
husbandry practices that mitigate important environmental risk factors,
and as knowledge of the molecular features, detection methods and
options for AHPND management have evolved.
The etiologic agent of the disease was originally identified as Vibrio
parahaemolyticus (Tran et al. 2013, Han et al. 2015b). The plasmid
containing the pirAB genes that code for binary PirAB toxins in V.
parahaemolyticus is now known to be the main virulence factor of this
disease (Han et al. 2015a, Sirikharin et al. 2015). The list of bacteria
that can cause AHPND include: Vibrio campbellii (Han et al. 2017), Vi-
brio harveyi (Kondo et al. 2015), Vibrio owensii (Liu et al. 2018) and
Vibrio punensis (Restrepo et al. 2018). The pathology of AHPND has two
distinct phases. In the acute phase, the infected hepatopancreas shows
detachment of tubule epithelial cells from the basement membrane and
tubule epithelial degeneration in the absence of bacterial cells. In the
terminal stage, the hepatopancreas shows extensive intra-tubular he-
mocytic infiltration and the development of massive secondary bac-
terial infection (Tran et al., 2013).
In Latin America, there is strong evidence that AHPND is present in
Mexico (Nunan et al, 2014, Belize (UNCTAD, 2018), Costa Rica, Hon-
duras (Jun et al., 2016; Peña et al., 2020), as well as in South American
countries (Restrepo et al., 2018). Even though the Latin American strain
of VPAHPND is also highly pathogenic (Nunan et al., 2014), VPAHPND
does not cause acute mortalities and, in countries such as Ecuador and

⁎
Corresponding author at: School of Animal and Comparative Biomedical Sciences, The University of Arizona, Tucson, AZ 85721, USA.
E-mail address: lfarangu@email.arizona.edu (L.F. Aranguren Caro).

https://doi.org/10.1016/j.jip.2020.107424
Received 13 March 2020; Received in revised form 4 June 2020; Accepted 5 June 2020
Available online 11 June 2020
0022-2011/ © 2020 Elsevier Inc. All rights reserved.
L.F. Aranguren Caro, et al.
Peru that are the major shrimp producing countries in the region,
production has increased steadily since 2001 (Piedrahita, 2018; FAO,
2018).
The purpose of the present study was to explain the phenomena
observed at farm level using Penaeus vannamei shrimp lines from Latin
America in VPAHPND challenges using APL bioassay trial methodology.
Additionally, assessments were performed to document histopathology,
hepatopancreas heterotrophic plate count (HPC) and pirAB gene ex-
pression in shrimp sampled from the challenge studies.
2. Materials and methods
2.1. Shrimp and inoculum
Latin American lines of P. vannamei (APE) were imported into the
United States several years ago by a commercial operation. In
2017–2018, three lines of fifth generation offspring derived from the
founders were submitted to the Aquaculture Pathology Laboratory
(APL) at University of Arizona for a VPAHPND challenge bioassay. Since
entry into the US, the imported shrimp and their offspring were routi-
nely tested by APL for OIE-listed pathogens and test results were ne-
gative for the OIE-listed pathogens including VPAHPND and EHP.
Specific-pathogen-free (SPF) P. vannamei, known to be highly suscep-
tible to AHPND, were supplied by a commercial entity in the US and
served as the positive control line in the two trials.
The reference strain, V. parahaemolyticus, 13-028/A3 (designated
VPAHPND), an AHPND causing bacterial strain (Tran et al., 2013), was
used for both challenge trials. VPAHPND was grown in tryptic soy broth
with 2% NaCl (TSB+) at 28–29 °C with shaking (120 rpm) for 18 h or
until a bacterial optical density absorbance of 3.0 at OD600 nm (~109
CFU/ml) was achieved
2.2. Challenge tests
Two VPAHPND challenge trials were conducted for this study. All
tanks in the trials were similar: circular, 1.81 m2 bottom surface area,
volume of 1000 L; and individual tanks were outfitted with a pre-ac-
climated biological filter and adequate aeration. All tanks were covered
with a plastic sheet to reduce the risk of cross contamination. Shrimp in
all tanks were fed once daily with a 40% protein concentration com-
mercial pelleted ration at 5% estimated shrimp biomass. VPAHPND in
TSB was added directly into the water of each challenge tank (in both
trials) to achieve an estimated density of 1 × 105 CFU/ml of the
bioassay tank water. Water was not exchanged after the bacterial in-
oculation. Mortality was recorded daily for each tank from the start of
each experiment.
2.2.1. Trial I:
The purpose of Trial I was to compare survival of the three in-
oculated shrimp lines, the negative controls and the SPF positive con-
trol. Two SPF tanks and three tank replicas for each of the test lines
were subjected to immersion challenge. The number of shrimp in each
tank was 21 ± 3. The SPF population in the holding tanks were con-
sidered as the negative control treatment test for the SPF line in Trial I
(Table 1).
Water quality (temperature, salinity, pH, total ammonia nitrogen
(TAN) and nitrite) measurements were done using a hydrometer, pH
strips (VWR) and API ammonia test kit® respectively. The measurement
values ranged: temperature: 26-28℃, salinity: 26 −30‰, pH: 8.0, TAN:
0 – 0.5 ppm and nitrite: 0 – 0.5 ppm. Moribund shrimp were fixed in
Davidson’s alcohol-formalin-acetic acid (AFA) fixative (Bell and
Lightner, 1988). Samples of dead shrimp were collected to determine
the presence of VPAHPND by PCR. All surviving shrimp were fixed at the
end of the challenge for histology examination. The duration of the
assay was 11 days.
2
Journal of Invertebrate Pathology 174 (2020) 107424
2.2.2. Trial II:
Trial II was conducted to measure hepatopancreas (HP) hetero-
trophic plate count (HPC), pirAB gene expression, histology at 24 h
post-infection (hpi), as well as final survival count and histology at trial
termination 6 d post infection (dpi) To compensate for a possible tank
effect, seven shrimp from each line and a similar number of SPF po-
pulation were placed into one 1000-L tank, whilst remaining tagged
shrimp from each line were placed into the negative control tank. To
identify individual shrimp to line, each animal was injected with a
colored elastomer tag, a different color for each line (Northwest Marine
Technology, Inc.) (Table 2).
On day 0, the treatment tank was VP AHPND-challenged following the
protocol described for Trial I. At approximately 24 hpi, two live shrimp
from each line were selected at random from the two tanks and the
shrimp were sacrificed one at a time, dissected and the hepatopancreas
removed for sample collection. Each hepatopancreas was divided
(longitudinal sections) into three approximately equal pieces and each
portion was weighed. One piece was preserved in 95% ethanol and later
evaluated by qRT-PCR for pirA and pirB gene expression, another piece
was preserved in Davidson’s fixative for histological analysis and the
remaining tissue aliquot was macerated, serially diluted in sterile saline
and inoculated onto Trypticase Soy Agar+2% (TSA+) sodium chloride
to determine the CFU/g of the HP tissue sample.
2.3. Duplex PCR for the detection of pirA- and pirB-like genes
For the duplex PCR assay to detect pirA and pirB, PuReTaq ready-to-
go PCR beads and the primers VpPirA-284F TGA CTA TTC TCA CGA
TTG GAC TG /R VpPirA-284F TGA CTA TTC TCA CGA TTG GAC TG
that amplify a region of 284 bp and VpPirB-392F TGA TGA AGT GAT
GGG TGC TC /R VpPirB-392R TGT AAG CGC CGT TTA ACT CA that
amplify a region of 392 bp were used. Amplifications were performed
with the following parameters: initiation denaturation: 94 °C for 3 min,
followed by 35 cycles at 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s,
and a final extension at 72 °C for 7 min (Han et al., 2015a). Following
quantitative PCR (qPCR), an aliquot of PCR products was analyzed in a
1.5% gel containing 1X gel red (Biotium, US).
2.4. RNA extraction & pirAB gene expression analysis
RNA was extracted individually from each hepatopancreas using
RNAzol kit (MRC, USA). RNA was treated with DNase 1 (Invitrogen,
USA). Primer express software (Applied Biosystems) was used to design
the forward and reverse primers to amplify pirA and pirB genes from
AHPND-causing V. parahaemolyticus (GenBank KM067908): PirAF (5′-
CAA ACG GAG GCG TCA CAG A-3′), and PirAR (5′ GAC CGA CTT CCG
GGA TGA T-3′), PirBF (5′- TGC AAA CCA AGA TAA CGT GTA TGA-3′),
and PirBR (5′ GCC GTG AAC CGT ACA CCA A-3′). Amplification was
carried out in a final volume of 20 μl with 10 µl of PowerUP SYBR
Green Master Mix 2X, 0.4 μM of each primer and 2 µl of RNA. The
reverse transcriptase qPCR (RT-qPCR) method consisted of 2 min at
50 °C and 2 min at 95 °C followed by 40 cycles of 3 s at 95 °C and 30 s at
60 °C and dissociation step (Fig. 5). The amplicon product was 60 bp for
pirA and 64 bp for pirB, respectively. The Ct value of each gene was
normalized to the Ct value of the housekeeping gene elongation factor1-
alpha (EF1-α) (Dhar et al., 2002).
2.5. Histopathology
The Davidson's (AFA)-fixed samples were processed, embedded in
paraffin, and sectioned (4 μ thick) in accordance with standard methods
(Bell and Lightner, 1988). After staining with hematoxylin and eosin (H
&E), the sections were analyzed by light microscopy. Severity grade of
infection/lesion ranged from G0-G4 according to Lightner (1996); G0
being absence of the disease and G4 indicating presence of severe le-
sions and advanced tissue destruction.
L.F. Aranguren Caro, et al. Journal of Invertebrate Pathology 174 (2020) 107424

Table 1
Description of the shrimp lines and treatments utilized in the VPAHPND challenge, Trial I.
Tank Number Shrimp Line Number of shrimp Treatment Immersion Challenge Dose of AHPNDVP (CFU/ml)

A4 APE2 22 AHPND Negative control Unexposed

A5 APE3 19 AHPND Negative control Unexposed
A6 APE1 25 AHPND Negative control Unexposed
L1 SPF 20 AHPND Positive control 1 × 105
L2 SPF 20 AHPND Positive control 1 × 105
D1 APE2 18 AHPND 1 × 105
D2 APE2 19 AHPND 1 × 105
D3 APE2 19 AHPND 1 × 105
D4 APE3 19 AHPND 1 × 105
D5 APE3 21 AHPND 1 × 105
D6 APE3 17 AHPND 1 × 105
5A APE1 25 AHPND 1 × 105
5B APE1 28 AHPND 1 × 105
5C APE1 25 AHPND 1 × 105

Table 2 3.2. PCR**a

Distribution of tagged shrimp in the VPAHPND-challenged tank in Trial II.
Tank Shrimp Line Number of Immersion Challenge Dose of
Eleven samples of hepatopancreas of dead shrimp in the VPAHPND
Number Shrimp AHPNDVP (CFU/ml) Trial I were collected and pooled per line/tank assayed by duplex PCR
to detect pirA and pirB. All pools were positive for both, pirA and pirB
1 APE1 7 1 × 105 and the bands that amplify the region of 284 bp and 392 bp were
APE2 7 1 × 105
APE3 7 1 × 105
clearly observed. Pools from the unexposed lines (negative controls)
SPF 21 1 × 105 were pooled per tanks and assayed by duplex PCR with negative results
3 APE1 6 AHPND Negative control in all cases.
APE2 4
APE3 7
SPF 17
3.3. Histopathology

2.6. Bacteriology The histopathology of the SPF population in Trial II is presented in

For bacterial count, the third section of the HP was placed into a
microtube immersed in ice. Each HP was weighed and placed in Tryptic
Soy Broth (TSB) + 2% sodium chloride and the homogenate was 10-
fold diluted from 1x10-1 to 1 × 10-5. Using dilutions 1 × 10-3, 1 × 10-4
and 1 × 10-5, samples were inoculated onto Trypticase Soy Agar
(TSA) + 2% sodium chloride plates using the streaking method, and
incubated for 24 h at 29–30 0C. At 24 h, HPC was determined and the
colony forming units (CFU) recorded for each sample.
2.7. Statistical analyses
Survival curves were analyzed by Kaplan-Meir survival analysis
using the software Med Calc. 16.8.4. Between and within lines (control
vs. treatment) and treatment vs. control groups, mean values for mea-
surement data sets were compared by two sided t-test (α = 0.05) or one
way ANOVA using StatView®.
3. Results
3.1. Cumulative survival
3.1.1. Trials I and II
Analysis of the survival values using Kaplan-Meier (Fig. 1) revealed
a significant difference (P < 0.001) between APE1 (72.1 ± 17.4%) and
the three other lines exposed to VPAHPND.
APE2 & APE3 had similar final survival of 16.1 ± 5.3% and
17.0 ± 10.1%, respectively, and the SPF line had the lowest survival
(12.5 ± 10.6%). Survival was not significantly different (P > 0.05)
between APE2, APE3 and SPF. The mean negative control survival
(95.2 ± 4.6%) of each of the four lines was significantly higher
(P < 0.001) than the mean survival of the corresponding treatment.
Fig. 2 and Fig. 3. The Davidson’s-fixed healthy shrimp from the SPF line
negative control showed normal structure of tubules and epithelial cells
in the hepatopancreas including a high level of lipid droplets (R-cells),
secretory vacuoles (B-cells) and absence of AHPND (Fig. 2A). In con-
trast, SPF shrimp fixed in the first and second dpi displayed lesions
typical of VPAHPND in acute phase, including a multifocal necrosis and
massive sloughing of HP tubule epithelial cells in the medial region of
the hepatopancreas and progressing outward to the distal region. Very
low levels of B-cells and R-cells were observed (G0-G1) and hemocytic
infiltration surrounding necrotic tubules was common. At this point,
bacterial colonization was not observed (Fig. 2B). Other SPF shrimp
analyzed displayed the typical VPAHPND terminal phase characterized
by necrosis and sloughing of HP tubule epithelial cells (Fig. 2C) to
massive bacterial presence and host inflammatory response between
and within HP tubules. Interestingly, some survivors at 7 dpi showed
only a few tubules with epithelial necrosis accompanied by bacteria and
inflammation, which resembles a septic hepatopancreatic necrosis
(SHPN) (Fig. 2D). This had not previously been described as a type of
lesion in the case definition for AHPND.
The histopathology of shrimp representing the Latin American lines
(APE1, APE2 & APE3) is presented in the Fig. 3. Fig. 3A shows the HP
histology of a healthy shrimp from the APE negative control tank. The
histology was similar to the normal HP histology in the animals from
SPF line. Fig. 3B and 3C, respectively, are examples of the typical acute
and terminal phase of AHPND and. Fig. 3D is an example of the ap-
pearance of chronic phase AHPND histology for a survivor shrimp after
6 dpi. Similar chronic lesions were observed in shrimp in the SPF line.
The granulomatous response in the HP tubules is an indicator of an
inflammatory response at a more advanced stage of maturity in chronic
phase in AHPND-affected shrimp. Fig. 3E presents a suspected case of
AHPND (G-trace) with focal necrosis and sloughing of tubules epithelial
cells in the medial portion of HP. Two survivors from APE1 had no
histopathological lesions indicative of AHPND, which suggests a pos-
sible tolerance/resistance to AHPND infection (Fig. 3F) Table 3.

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L.F. Aranguren Caro, et al. Journal of Invertebrate Pathology 174 (2020) 107424

Fig. 1. Trial I: Cumulative survival for four Penaeus

100.0
vannamei lines exposed to VPAHPND (1 × 105CFU/
ml). Negative control (NC) for APE1-3 (red solid
90.0
line), APE1 (dashed blue line), APE2 (dash-dot
line), APE3 (dotted line) and SPF (black solid line).
80.0
Data shown are the average of three (APE1, 2, 3) or
two (SPF) replicates. Error bars represent the 95%
70.0
confidence interval of the data set.
60.0
Cumulative survival (%)

50.0

40.0

30.0

20.0

10.0

0.0
0 1 2 3 4 5 6 7 8 9 10 11
Days post infection

NC SPF APE1 APE2 APE3

3.4. Bacteriology challenged cohort samples (2.13x107 CFU/ g HP) in the study.

The HPC for the HP of control and VP AHPND-challenged shrimp at 24
hpi are summarized in Fig. 4. For the negative control group, the
highest mean count was recorded for APE1 (2.22 × 107 CFU /g HP). In
contrast, the lowest mean control count was for the SPF line
(1.05 × 104 CFU /g HP). The 24 hpi VPAHPND challenge tank samples
showed that the HPC increased significantly (P < 0.05) between con-
trol and treatment shrimp for APE2, APE3 and the SPF line; with the
largest differential mean count, 3.70 × 107 CFU/ g HP, for the SPF line,
~103 times higher than the mean negative control count for this line
(1.05 × 104 CFU/ g HP). APE1 had little change in mean CFU /g HP
between the negative control (2.20x107 CFU/ g HP) and the VPAHPND
3.5. Gene expression analysis
The quantitative load of pirAB transcripts in hepatopancreas tissue
of VPAHPND-challenged animals (SPF and APE lines) was measured by
RT-qPCR. Table 4 shows the normalized Ct values of pirA and pirB in the
different shrimp lines. No significant mean difference (P > 0.05) was
found in pirAB gene expression at the transcription level among the SPF
and APE lines examined. There was no correlation amongst HP bac-
terial count and Ct value for individual shrimp sampled in the study nor
between H&E grade of infection and CT values in any of the four lines
(Table 4).

Fig. 2. H&E (Mayer–Bennet hematoxylin and

eosin-phloxine) histology of VPAHPND-infected
SPF shrimp at 24 hpi-6 dpi: (A) P. vannamei
from the negative control tank (B) Acute phase
of AHPND infection (C) terminal phase of
AHPND infection (D) Chronic phase of AHPND
infection. Scale bars = 100μ. Photos B – D
represent specific-pathogen-free line shrimp.

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L.F. Aranguren Caro, et al. Journal of Invertebrate Pathology 174 (2020) 107424

Fig. 3. H&E (Mayer–Bennet hematoxylin and eosin-phloxine) histology of VPAHPND-infected APE shrimp at 24 hpi-6 dpi: (A) Penaeus vannamei from the negative
control tank (B) Acute phase of AHPND infection (C) terminal phase of AHPND infection (D) Chronic phase of AHPND infection. (E) Possible AHPND G-trace. (F) Line
1 survivor shrimp challenged with VPAHPND sampled at 6 dpi. Scale bars = 100 μm. Photos B – F represent APE lines shrimp.

Table 3 lesions were recorded in moribund shrimp in the two trials. In Trial I,
Trial II. The number of shrimp with AHPND lesions after VPAHPND experimental the surviving shrimp (6 dpi) also showed histological changes con-
infection at 24 hpi and 6 dpi. sistent with septic hepatopancreatic necrosis (SHPN) (Fig. 2D, 3C &
Treatment APE1 APE2 APE3 SPF 3D). It is interesting to note that SHPN was not included in the pa-
thological description of AHPND (Lightner and Flegel, 2012; OIE,
Negative control tank 0/2 0/1 0/1 0/4 2018), probably because during the first years of the infection in SE
AHPND infected tank 1/2 (G1) 1/2 (G4) 1/2 (G4) 4/4 (G2-G3)
Asia, mortality was close to a 100% in most cases. To our knowledge,
24 h.p.i
AHPND infected tanks 6 0/2 2/2 (G2) 1/1 (G4) 1/2 (G2) this is the first record of SHPN in VP AHPND-infected shrimp. Other
d.p.i Chronic Chronic Chronic chronic, hepatopancreas changes observed were granulomata, focal to
multifocal melanization of HP tubules, low cytoplasmic lipid and
atrophy of tubule epithelial cells. One survivor from the APE1 group
4. Discussion had mild tubule epithelial necrosis (Fig. 3E) and, in another specimen,
no AHPND lesions were observed at all (Fig. 3F). These histological
In this study, four P. vannamei shrimp lines, including an SPF and features are suggestive of tolerance/resistance in shrimp from APE1 line
three Latin American shrimp lines, were challenged via immersion with (see Table 3).
a virulent strain of VPAHPND using a bacterial cell density of ~1 × 105 In Trial II, the HPC in hepatopancreas increased at 24 hpi in the
CFU/ ml. This was a similar dosage of VPAHPND that was recently re- VPAHPND treatments compared to the negative control tank for the
ported in challenge studies aimed at detecting differences in survival APE2, APE3 and the SPF lines. The CFU/ g HP control counts in APE2,
among genetically unrelated lines of P. vannamei shrimp (Soto- APE3 and SPF ranged ~104 to ~106; whereas for APE1 the CFU/ g HP
Rodriguez et al. 2015; Aranguren et al., 2017). One of the Latin were significantly higher (mean = 2.20 × 107 g HP). The highest
American lines (APE1 line) had significantly higher survival than the control to challenged differential was 1: 3.01 × 103 for the SPF line;
APE2, APE3 and the SPF lines to VPAHPND challenge under our ex- whereas for APE1, the mean difference was 1.04:1, essentially
perimental laboratory conditions. AHPND acute and terminal phase

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L.F. Aranguren Caro, et al. Journal of Invertebrate Pathology 174 (2020) 107424

Fig. 4. Comparison of the heterotrophic plate count (HPC) determined in CFU g − 1 HP in four lines of shrimp exposed with VPAHPND at 24 hpi. NTC: Negative
control; AHPND: challenge with VPAHPND. Data represent the mean ± SE.

Table 4 This is well below the mean CFU measured for APE1 pre-challenge
The mRNA expression of pirA & pirB genes in hepatopancreas of VPAHPND control sample in the study. However, TCBS is a selective media;
challenged SPF and Latin American lines of Penaeus vannamei. Data represent whereas TSA is not; thus, a comparison of count data between this study
the quantification threshold (Ct) after normalization ± Standard deviation and the earlier work should take this difference into consideration.
(SD). Following VPAHPND challenge, the mean CFU/ g HP increased for
Line pir A pir B APE2, APE3 and SPF. The mean HPC was 1.7 × 107 CFU g HP-1 (range:
9.52 × 104 – 1.05 × 108 CFU g HP-1). Although the bacterial load was
Mean Ct SD Mean Ct SD
quite variable among different animals, the CFU mean was consistent
APE 1 9.70 1.05 9.05 3.73 with previous challenge test studies done in APL (Aranguren et al.,
APE 2 10.39 5.91 13.94 6.07 2017). In this study, the hepatopancreas bacterial count was comprised
APE 3 5.43 0.30 8.71 0.95 of the VPAHPND as well as the natural bacterial flora and likely increased
SPF 6.79 3.38 9.60 2.27 in response to the increased availability of nutrients (necrotic cellular
debris and exposed tubule basal membrane) in the affected HPs.
Oanh et al. (2016) demonstrated reduced VPAHPND impact in test
unchanged between the control and VPAHPND-challenged shrimp. As a
groups receiving probiotic treatments compared to the positive control
point of reference, Gomez-Gil et al. (1998) reported bacterial density in
(no probiotic added). In a more recent work, Pinoargote et al. (2018)
HP of healthy, P. vannamei as mean = 4 × 104; range 102 to 105 CFU/ g
reported that adding several strains of probiotic bacteria (including
HP, which is quite similar to our data in the unexposed APE2, 3 and SPF
lactic acid bacteria combined with yeast and photosynthetic bacteria)
lines. In those lines, CFU/ g HP count varied from 1.05 × 104 to
into the tank water (108 – 109 CFU/ ml) and feed for 7 d prior to
8.15 × 105 CFU g HP, which is within the range reported previously.
AHPND challenge (dose 106 CFU ml tank water) resulted in delay in
Soto Rodriguez et al. (2010) reported bacterial counts of
onset of mortality and a significantly increased final survival rate
1.40 ± 3.20 × 105 CFU/ g HP using TCBS medium for farmed P.
compared to the positive control group exposed to VPAHPND alone. In
vannamei submitted for diagnostics between 2001 and 2006 in Mexico.

Fig. 5. Schematic of the process of the mRNA expression of pirA & pirB genes.

6
L.F. Aranguren Caro, et al.
the present study, higher survival was associated with very high he-
patopancreas HPC prior to VPAHPND challenge in APE1. Perhaps, an
initially high bacteria biomass in the stomach/hepatopancreas had a
negative influence on V. parahaemolyticus colonization and/or replica-
tion, either through direct bacteria to bacteria antagonism and/or the
association of higher bacterial load with greater activity of the shrimp
innate immune system, thus, providing the individual animals in APE1
an increased tolerance to AHPND. In any case, the reason(s) behind the
higher survival recorded for APE1 would be worthwhile to explore
further.
The expression of pirA and pirB genes at 24 hpi was demonstrated in
HP samples from shrimp in the four lines. However, there was no sig-
nificant difference in pirA and pirB expression among those lines, sug-
gesting that there was not a correlation between HP CFU and Pir AB
gene expression. Similarly, Tinwongger et al. (2016) reported no cor-
relation between expression of pirAB genes and copy number of plas-
mids.
Despite the strong evidence of the presence of AHPND in Latin
America (Nunan et al., 2014; Jun et al., 2016; Peña et al., 2020;
Restrepo et al., 2018), shrimp production in this region has grown
continuously over the past 10 years, (FAO, 2018). The chronic mor-
talities caused by AHPND at the farm level in grow-out ponds were
similar to those reported in this study of VPAHPND challenge test of the
shrimp lines used in Latin America. In order to explain this AHPND
resistance/tolerance, it is important to consider the history of the
shrimp industry in Latin America. Since the early days, the vast ma-
jority of farms in Latin America have utilized a “live with” the patho-
gens approach, referred to as All Pathogens Exposed (APE).
The shrimp industry in Latin American countries has been exposed
to several pathogens during the last 30 years including viral pathogens
such as Taura syndrome virus (TSV) during 1992–1995 (Hasson et al.,
1995, Brock et al., 1995) and WSSV since 1999 (Lightner 1999). With
no further options, some of the major producers initiated a program to
select the survivors at the beginning from TSV-infected ponds and later
on from WSSV-infected ponds (Cock et al. 2009). In the mid-term, the
shrimp industry began seeing some recovery in ponds with previous
infections by these viral pathogens. From 2010 to 2019, the shrimp
industry in Ecuador, by far the largest shrimp producer in the Americas,
has seen increased pond survival and production (Piedrahita 2018)
despite the presence of these pathogens in the culture ponds. During the
TSV and WSSV selection process by the shrimp farmers, some other
diseases, including bacterial diseases, have been present at the farm
level including, SHPN, Seagull syndrome (Lightner 1996), and some at
hatchery level, including bioluminescent V. harveyi and bolitas syn-
drome, which might suggest that these APE populations have been
exposed to several bacterial pathogens, acquiring some tolerance to
multiple pathogens simultaneously. (Aranguren et al., 2010) found that
shrimp in Colombia selected for resistance to TSV indirectly acquired
resistance to necrotizing hepatopancreatitis, an endemic disease present
in the broodstock ponds at that time. Similar phenomena could have
occurred with these Latin American stocks that encountered Vibrio in-
fection during the culture at farm conditions.
In summary, we have described bioassay challenge results that
identified one of three Latin American shrimp lines that displayed
higher survival (~70%) to AHPND challenge compared to two other
APE lines and the SPF positive control lines. The difference in survival
could be attributed to either the elevated numbers of bacteria recorded
in the HP of individual APE1 animals and/or as yet unknown resistance
factors to AHPND in this line. Additional comparative studies are
planned to learn more about qualitative and quantitative bacteriology
in hepatopancreas samples collected in Trial II.
Acknowledgements
The authors would like to thank Dr. James Brock for reviewing
critically the manuscript and make valuable comments to improve it &
7
Journal of Invertebrate Pathology 174 (2020) 107424
Jasmine Millabas for providing technical assistance. Partial funding for
this work is supported by the USDA National Institute of Food and
Agriculture, Animal Health project 1006512 to AKD.
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