Animal Feed Science and Technology 297 (2023) 115581

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Animal Feed Science and Technology

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

Dietary montmorillonite clay improved Penaeus vannamei survival

from acute hepatopancreatic necrosis disease and modulated
stomach microbiota
Wing-Keong Ng a, b, *, Mei-Ling Mong b, Abdul-Azim Abdul-Hamid b
a
Asian Aquafeeds Services, 1727 Lavinia, Taman Sri Nibong, 11900 Penang, Malaysia
b
School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia

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

Keywords: Acute hepatopancreatic necrosis disease (AHPND) is causing a bacterial pandemic in shrimp
Montmorillonite clay farming. AHPND is caused by pathogenic strains of Vibrio parahaemolyticus (VPAHPND) that pro­
Penaeus vannamei duces the bacterial toxins, PirA and PirB. Shrimp exposure to AHPND-causing bacteria is hy­
AHPND
pothesized to be through the oral route, ingested into the gut where the bacteria initially
Microbiota
Functional feeds
colonized the stomach and then releases the binary toxins to damage the hepatopancreas. In view
of changing global regulatory controls on the use of antibiotics, there is much interest in the
development of functional aquafeeds. One innovative method to mitigate AHPND is to bind the
bacterial toxins by adding adsorbent clay minerals into shrimp feeds. A feeding trial using trip­
licate groups of Penaeus vannamei post-larvae (PL30) was conducted to evaluate the efficacy of a
montmorillonite (MMT) clay [Calibrin®-Z (CL)] on growth, gut health and disease resistance to
AHPND. The addition of 0.25% or 0.50% CL did not significantly (P > 0.05) impact growth and
feed utilization efficiency compared to control groups fed diets with 0% CL. When challenged
with VPAHPND, survival of shrimp fed 0.25% or 0.50% CL were 83.3 ± 5.5% and 93.8 ± 0.1%,
respectively, and were significantly higher (P < 0.05) compared to the VPAHPND-challenged
positive control group (39.6 ± 10.4%) but not significantly different compared to the unchal­
lenged negative control group (95.8 ± 2.1%). Vibrio and total cultivable bacteria counts in the
hepatopancreas of shrimp fed CL-added diets were significantly lower compared to positive
control. Hepatopancreas histopathology of infected shrimp fed CL-added diets showed less
damage. During AHPND infection, bacterial diversity was depressed but dietary CL tended to
restore stomach bacterial richness and α-diversity index. Dietary CL modulated the stomach
bacterial community possibly with beneficial impact to shrimp survival. At the phylum level,
Proteobacteria and Bacteroidetes were the main groups but the relative abundance of Verruco­
microbia, Acidobacteria and Firmicutes was more prevalent in the microbiota of shrimp fed CL-
added diets. Bacteria of the genus Pseudoalteromonas, Tenacibaculum, and Marinimicrobium were
identified as members of the healthy microbiota and Lysobacter was observed to be relatively
enriched in AHPND-infected shrimp. Demequina was identified as a potential biomarker and was
significantly enriched in the stomach of the CL-added groups. This is the first report on the
effectiveness of dietary clay mineral in AHPND mitigation. At least 0.25% CL is suggested to be

Abbreviations: AHPND, acute hepatopancreatic necrosis disease; VP, Vibrio parahaemolyticus; PL, post-larvae; Pir, Photorhabdus insect-related;
MMT, montmorillonite; CL, Calibrin®-Z.
* Corresponding author at: Asian Aquafeeds Services, 1727 Lavinia, Taman Sri Nibong, 11900 Penang, Malaysia.
E-mail address: wkng.usm@gmail.com (W.-K. Ng).

https://doi.org/10.1016/j.anifeedsci.2023.115581
Received 13 October 2022; Received in revised form 9 January 2023; Accepted 20 January 2023
Available online 21 January 2023
0377-8401/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
W.-K. Ng et al. Animal Feed Science and Technology 297 (2023) 115581

included in commercial shrimp feeds to mitigate against production and economic losses from
AHPND outbreaks in shrimp farms.

1. Introduction

The Pacific white shrimp or whiteleg shrimp (Penaeus vannamei) is the most produced farmed shrimp species. Although not
indigenous to Asia, it is currently mainly produced (about 80%) by Asian countries such as China, Thailand, Vietnam, Indonesia and
India. The world aquaculture production of P. vannamei has achieved over 5.8 million tonnes in 2020 (FAO, 2022). Global exports of
shrimp and prawn represents a major source of foreign exchange earnings for many developing countries in Asia and Latin America
and were valued at USD 24.7 billion in 2020 (FAO, 2022). Increasing intensification of shrimp culture systems with higher stocking
densities, improved shrimp stocks, increased water input, artificial aeration and manufactured feeds has contributed to the increased
global production. With increasing intensification of culture systems, outbreaks of shrimp diseases are prevalent (Thitamadee et al.,
2016). In recent years, shrimp cultivation has suffered significant economic losses due to the emergence of a bacterial disease called
acute hepatopancreatic necrosis disease (AHPND) which had caused and is still causing shrimp mortalities from 40% to 100% during
the first 35–45 days after stocking shrimp post-larvae in grow-out ponds. It was first detected in China (2009) and spread sequentially
to Vietnam (2010), Malaysia (2011), Thailand (2012) (Tran et al., 2013; Thitamadee et al., 2016), Mexico (2013) (Nunan et al., 2014),
the Philippines (2014) (Dabu et al., 2017), South America and South Korea (2016) (Restrepo et al., 2016; Hwang et al., 2020),
Bangladesh and USA (2017) (Eshik et al., 2017; Dhar et al., 2019) and possibly beyond. It is speculated that AHPND is currently present
in the shrimp farming communities of more countries but not reported. An infectious disease spreading across countries and conti­
nents, AHPND is a bacterial pandemic in the shrimp farming world. Outbreaks of this disease has resulted in drastic production losses
and within a 6-year period between 2010 and 2016, between USD 1.3–11.0 billion were lost in many Asian shrimp industries, mainly
due to AHPND (Shinn et al., 2018).
AHPND-affected shrimp show signs of anorexia, lethargy, slow growth, a pale atrophied hepatopancreas, as well as an empty
gastrointestinal tract (Tran et al., 2013). The unique histological diagnostic characteristic of the disease is sloughing of the affected
shrimp hepatopancreas tubule epithelial cells into the tubule lumen at the early stage, while at the later stages it includes hemocytic
infiltration and massive secondary bacterial infection (Tran et al., 2013; Thitamadee et al., 2016). In the year 2013, the causative
vector of AHPND was identified as a unique strain of the bacteria Vibrio parahaemolyticus (Tran et al., 2013). These pathogenic strains
carry a unique pVA-type plasmid which has Photorhabdus insect-related (Pir) genes that produces a binary toxin (Pir A and Pir B) which
causes the destruction of the shrimp hepatopancreas (Han et al., 2015a). Subsequently, it was found that AHPND can also be caused by
pathogenic strains of other Vibrio species including V. harveyi, V. campbellii, V. punensis and V. owensii, if they harbor the transferable
pVA-type plasmid (Kondo et al., 2015; Kumar et al., 2020). It has been hypothesized that shrimp exposure to AHPND-causing bacteria
is through the oral route, ingested into the digestive tract where the bacteria initially colonized the stomach and then releases
PirAB-like toxins to damage the hepatopancreas (Kumar et al., 2020). The pathogenesis of AHPND is currently an active evolving
science of discovery.
Bacterial diseases such as AHPND continue to be a problem and limit the productivity of many shrimp farms, leading to antibiotic
use as a prophylactic and/or treatment during disease outbreaks. The overuse and misuse of antibiotics is prevalent in Asia. This
practice can lead to increased antibiotic resistance in the pathogens of shrimp, which can also be transferred to animal and human
pathogens, leading to an overall increase in infectious diseases. Studies have revealed that Vibrio bacteria causing AHPND have now
shown antibiotic resistance to a wide range of antibiotics (Lai et al., 2015) including plasmid-mediated tetracyline resistance in
V. parahaemolyticus (Han et al., 2015b). With changing global regulatory trends on the use of antibiotics in animal farming, it is
imperative that alternatives to the prophylactic and therapeutic use of antibiotics in shrimp aquafeeds be found. Oral administration of
functional feed additives such as prebiotics, probiotics, organic acids and phytogenics are currently being explored as potential al­
ternatives to antibiotics to enhance shrimp growth and resistance to AHPND (Soowannayan et al., 2019; Koch et al., 2020; Kumar et al.,
2020). These feed additives depend on various modes of action (eg. immune boosting, antimicrobial, quorum quenching, etc) directly
or indirectly targeting the Vibrio bacteria and/or shrimp host. An innovative idea in mitigating AHPND infection would be to directly
bind the PirA and PirB toxins resulting in un-absorption from the digestive tract and the toxins being removed from the body via fecal
expulsion before they can migrate to the hepatopancreas to cause damage. Clays (bentonites) and clay minerals (eg. montmorillonite)
are commonly used as adsorbents to remove toxic mycotoxins from animal feeds (Li et al., 2018). Leveraging on their existing
commercial calcium montmorillonite product (Calibrin®-Z) used as a mycotoxin adsorber in animal feeds, a company in USA (Amlan
International Inc.) is now also marketing it as a solution to AHPND. As far as we know, other than several oral presentations given at
conferences, there is currently no published report in scientific journals on the effectiveness and efficacy of Calibrin®-Z (CL) or any
other clay product to mitigate AHPND.
Montmorillonite (MMT) belongs to the smectite group of clay minerals and have the general formula of (Ca, Na, H)(Al, Mg, Fe,
Zn)2(Si, Al)4O10(OH)2.nH2O (Uddin, 2018). The exact chemical formula of MMT can vary due to the modifiable structure and chemical
composition but essentially, it is a hydrated calcium and/or sodium aluminosilicate. MMT is a clay mineral having a 2:1 sheet structure
of two silicon oxide tetrahedral layers and an aluminum hydroxide octahedral layer joined together by electrostatic and Van der Waals
forces, interlayer cations or by hydrogen bonding (Uddin, 2018). An important source of MMT in nature is bentonite rock of clay. The
main components of CL were reported to be at least 70% MMT and 15% amorphous hydrated silicon dioxide (Chen et al., 2020).
Bentonite and MMT clays are considered industrial minerals (Section L, E558) and are allowed as feed additives to a maximum of 20

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g/kg feed according to Annex 1 of Directive 70/524/EEC of the European Union (EU) authorities.
The use of MMT in animal feeds have been reviewed by Liu et al. (2021). MMT as an animal feed additive had been reported to be
effective in removing mycotoxins, heavy metals and bacteria in feed and gut of farmed animals by surface adsorption, electrostatic
adsorption and intermolecular force. Improvement in gut health, growth and product quality had been reported in livestock fed
MMT-supplemented feeds (Liu et al., 2021). Compared to poultry and pigs, very little information has been published on the use of
MMT in fish and shrimp feeds. Dietary MMT had been reported to be effective in mitigating the toxic effects of aflatoxin and heavy
metals in Nile tilapia Oreochromis niloticus (Dai et al., 2010; Zychowski et al., 2013; Mahrous et al., 2015). It acted as a growth pro­
moter, improved immune response and increased disease resistance against viral hemorrhagic septicemia virus in rainbow trout
Oncorhynchus mykiss (Karimi et al., 2020). In turbot Scophthalmus maximus, Shi et al. (2022) reported improved growth and gut health
with higher gut microbiota diversity. Zhang et al. (2022) reported that kuruma shrimp Marsupenaeus japonicus fed MMT-added diets
showed increased antioxidant enzyme activities and improved gut health. Detailed information of dietary MMT in improving the
health condition of farmed aquatic animals is scarce and the effectiveness of MMT in mitigating AHPND in P. vannamei has not been
previously reported in peer-reviewed journals.
The objective of this study was to assess if, and to what extent, dietary CL inclusion could provide benefits for the growth per­
formance, feed utilization and disease resistance of P. vannamei to AHPND. To the best of our knowledge, this is the first report of the
effects of dietary MMT on shrimp stomach microbiota during an AHPND infection. Considering the magnitude of the AHPND pandemic
and the great interest from shrimp farmers in finding a cost-effective solution, investigating MMT as a potential alternative to harmful
antibiotics is pertinent and crucial.

2. Materials and method

2.1. Experimental shrimp diets

Four practical diets were produced using a commercial shrimp post-larval feed (Post Larva II crumbles #902, Gold Coin Specialities
Ltd., Malaysia) as the base formulation. The commercial shrimp feed was ground into fine powder before being re-pelleted. To prepare
the experimental diets, dry materials were mixed thoroughly with water in a Hobart mixer. Then, the moist dough was screw-pressed
through a 2-mm die using a locally assembled feed pelleting machine (Liang Traco, Penang). The feed pellets formed were fan-dried,
ground using a blender into appropriate sizes for post-larval shrimp, and stored in airtight polyethylene bags at − 20ºC until used. The
positive (posCON) and negative (negCON) control diet consisted of the re-pelleted commercial shrimp feed with the addition of a pellet
binder (Pegabind®, Bentoli AgriNutrition Co., Ltd., Thailand) (Table 1). Only the shrimp fed the posCON diet was later challenged
with VPAHPND. Two other diets with the inclusion of 0.25% (0.25% CL) or 0.5% (0.5% CL) montmorillonite clay (Calibrin®-Z, Amlan
International Inc., USA) at the expense of α-cellulose were also pelleted. These dietary levels were selected based on the manufacturer’s
recommendation for shrimp feeds. The control diets were without any added clay. The manufacturer’s advertised nutrient content (on
feed bag) of the commercial shrimp feed for crude protein, crude lipid, crude fiber, ash and moisture content was 38% (min), 7% (min),
3% (max), 15% (max) and 12% (max), respectively.

2.1.1. Feed pellet water stability

Approximately 2 g of representative feed sample of similar size (re-pelleted control diet and original commercial shrimp post-larval
feed) in triplicates were used for water stability analysis. The feed samples were placed in 250 ml-flasks filled with 100 ml artificial
saltwater (25‰ salinity, 28ºC) and placed on a INFORS HT Ecotron incubator shaker at 100 rpm shaker speed for the predetermined
immersion time (30, 60, 90, 120, 150, 180, 210 or 240 min, respectively). After immersion, the feed sample was gently rinsed with
distilled water to remove salt and recover all solids using a Buchner filtration apparatus with Whatman filter paper no. 3, followed by
further drying in the oven at 105ºC for 24 h and weighed. The percentage pellet stability in terms of dry matter retention was measured
for the corresponding time intervals using the following formula:

Dry matter retention (%) = [g feed remaining/g initial feed] x 100

From the test results, it was observed that experimental shrimp diet with added 0.8% Pegabind® as pellet binder imparted good

Table 1
Ingredient formulation of the experimental shrimp diets.
Montmorillonite clay (% of diet)

Ingredient (g/kg) 0 0.25 0.5

Shrimp feed (powdered)a 98.70 98.70 98.70

Calibrin®-Z montmorillonite clayb 0.00 0.25 0.50
Pegabind® binderc 0.80 0.80 0.80
α-cellulose 0.50 0.25 0.00
a
Post Larva II crumbles #902, Gold Coin Specialities Ltd., Malaysia. Disclosed proximate composition: crude protein, crude lipid, crude fiber, ash
and moisture content was 38% (min), 7% (min), 3% (max), 15% (max) and 12% (max), respectively.
b
Amlan International Inc., USA.
c
Bentoli AgriNutrition Co., Ltd., Thailand.

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water stability. The water stability of experimental re-pelleted shrimp diet ranged from 80.21% to 83.77% during the 4-h immersion
and were similar to that of commercial shrimp feed which ranged from 83.25% to 87.18%, with the highest percentage dry matter
retention observed after a 30 min immersion and decreasing thereafter.

2.1.2. Scanning electron microscopy

Calibrin®-Z was mounted onto aluminum stubs using adhesives. The clay specimens were then coated with gold using a Polaron
SC515 Sputter Coater and observed by a Scanning Electron Microscope [Leosupra 50vp Field Emission SEM equipped with Oxford
INCA 400 energy dispersive X-ray (EDX) microanalysis system]. SEM images were obtained for each clay sample. SEM was also used
for a preliminary mineral analysis of representative clay surfaces using the EDX microanalysis system.

2.2. Shrimp and feeding trial

The feeding trial was carried out at the Aquaculture Research Complex, School of Biological Sciences, Universiti Sains Malaysia,
Penang, Malaysia. The handling and use of shrimp were conducted according to guidelines from the USM Institutional Animal Care and
Use Committee, Universiti Sains Malaysia.
Specific pathogen free (SPF) post-larvae (PL8) of P. vannamei were procured from a local hatchery. After arrival at our facilities, all
shrimp were kept for two weeks in a fiberglass tank filled with 600 L of artificial seawater (Instant Ocean®, USA) at a salinity of 25 ±
1‰. During the acclimatization period, shrimp were fed to apparent satiation with a commercial shrimp feed crumble (Gold Coin
Specialities Ltd., Malaysia) three times daily. After two weeks, apparently healthy shrimp of similar size were randomly distributed
into a series of aquaria placed inside a bio-secure room and acclimated for one week with the control diet. The experimental aquaria
consisted of twelve 15-L aquariums equipped with a central air stone to provide continuous aeration. Plastic PVC tubes were provided
in each aquarium to act as shelters and increase the surface area to reduce cannibalism.
At the start of the feeding trial, all shrimp (PL30) were starved overnight, and groups of 20 apparently healthy shrimp were weighed,
transferred to each of the aquarium and randomly assigned to one of the dietary treatments. Triplicate groups of shrimp were hand-fed
with their respective diet three times daily at 09:00, 14:00 and 18:00 h to apparent satiation for two weeks. Water exchange of about
10% was done daily and fecal matter and leftover feeds siphoned out. All shrimp were counted and batch-weighed at the end of the
feeding trial.
Throughout the feeding trial, the salinity, dissolved oxygen, pH and temperature of culture water was within the range of 23 ± 1‰,
5.5 ± 0.1 mg/L, 8.1 ± 0.2, 28 ± 0.4 ◦ C, respectively.

2.3. AHPND bacterial challenge test

After the growth trial was completed, a sub-sample of the required number of shrimp from each dietary treatment were randomly
removed from each aquarium, pooled and then re-stocked at 16 shrimp per aquarium for the disease challenge trial. The disease
challenge was carried out using the immersion method (Tran et al., 2013) with slight modifications.
The disease challenge trial was conducted using V. parahaemolyticus (3HP strain), isolated from AHPND-positive shrimp (Centex
Shrimp, Thailand). To prepare the bacteria for immersion challenge, V. parahaemolyticus (VPAHPND) was activated from glycerol stock
and incubated in 30 ml of sterile tryptic soy broth plus 1.5% NaCl (TSB+) for 24 h at 37 ◦ C. The broth was then re-streaked on the
tryptic soy agar plus 1.5% NaCl (TSA+) plate and this was followed by placing a loop full of pure bacterial colonies into flasks con­
taining 150 ml of sterile TSB+ with vigorous shaking at 37 ◦ C for 24 h. After 24 h incubation, TSB+ suspensions were checked using a
spectrophotometer. An optical density (OD) at 600 nm of 0.6–0.8 was equivalent to a bacterial density of approximately 108 colony
forming units (CFU)/ml (Tran et al., 2013). The density of VPAHPND suspension was confirmed by standard plate count using TSA
plates.
Shrimp from each aquarium in the posCON, 2.5%CL or 5%CL groups were placed for 15 min (with aeration) in a 1-L Erlenmeyer
flask containing 150 ml of VPAHPND culture (108 CFU/ml). Then, the bacterial suspension including shrimp was poured directly back
into their respective aquarium containing clean artificial seawater to obtain a bacterial density of about 106 CFU/ml. This was similar
to the dosage of VPAHPND that was used in the challenge studies of Tran et al. (2013) and found to be effective. A negative control
treatment (negCON) was included to serve as an environmental control and the shrimp were not challenged with VPAHPND. Shrimp in
the negative control tank were fed with the control diet throughout and immersed in sterile TSB+ . During the 7-day disease challenge
trial, shrimp were fed two times daily and received a 50% water exchange in the morning. All shrimp were observed every three hours
to monitor gross clinical signs and mortality. Dead shrimp was recorded and removed from the experimental tanks.

2.4. Hepatopancreas microbiological analysis

The total cultivable bacteria count (TCBC) and presumptive Vibrio spp. count (PVC) in the shrimp hepatopancreas were determined
by direct plate counting (spread plate method) after the 7-day AHPND challenge. Briefly, three live shrimp were selected randomly
from each aquarium and the shrimps’ surface sterilized with 70% ethanol. The hepatopancreas were then aseptically dissected from
the rest of the gut taking care to avoid cross contamination, pooled, weighed and homogenized with sterile saline in serial dilutions
(10-fold). A volume of 100 µL aliquot of each dilution was spread plated in duplicates onto TSA+ and Thiosulfate-Citrate-Bile-Sucrose
(TCBS) agar plates and incubated at room temperature for 24 h for TCBC and PVC, respectively. Only plates that had between 30 and
300 colonies were used and the results expressed as log CFU/g.

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2.5. Hepatopancreas histopathology assay

Two moribund shrimp from each aquarium were collected when observed, and fixed in Davidson’s fixative for subsequent hep­
atopancreatic histological analysis. Sections were examined under a light microscope for typical AHPND lesions and AHPND infections
were confirmed by hepatopancreatic histology. The hepatopancreas were immersion fixed in Davidson’s solution for 24 h, processed
and embedded in paraffin wax. Section (5 µm thickness) were made using a rotary microtome and samples were stained with he­
matoxylin and eosin (H&E).

Fig. 1. Scanning electron microscopy micrograph of the montmorillonite clay, Calibrin®-Z. (A) shows the numerous stacked, latticed and inter­
connecting pores of the thermally-activated clay that has the ability to bind toxins (mag.12,000x). (B) shows the presence of opal lepispheres (OL)
which are micro-spherical aggregates of silicon dioxide, interspersed among the clay lattices (mag. 20,000x). Bars = 200 nm.

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2.6. Stomach microbiota analysis

2.6.1. Genomic DNA extraction

At the end of the disease challenge, three surviving shrimp were selected randomly from each treatment group and starved for two
days. Starvation was necessary to ensure collection of inhabiting microbiota of the stomach and not transitory microbes. Shrimp
stomach samples were aseptically dissected, taking care to avoid cross contamination and preserved in 1 ml of 99.7% ethanol before
genomic DNA extraction. After the removal of ethanol, the samples were resuspended in lysis buffer containing 100 mM Tris-HCL, 50
mM EDTA, 1% SDS and 200 µg Proteinase K followed by the addition of steel and silica beads for homogenization (TACO Prep Bead
Beater system, GeneReach, Taiwan) of the shrimp tissue and microbial cell wall. An overnight Proteinase K digestion at 55 ◦ C was
performed after homogenization. Protein precipitation was performed by the addition of saturated ammonium acetate followed by
incubation on ice for 15 min and centrifugation at 10,000 x g for 15 min. The supernatant was transferred to a new tube containing
isopropanol (1 x vol) and Sera-Mag™ carboxylate-modified magnetic particles (Cytiva, Brazil). After incubation at room temperature
for 10 min, the DNA-bound magnetic beads were separated from the solution followed by two rounds of 70% ethanol wash. The DNA
was eluted from the beads by the addition of 100 µ TE buffer and incubation at room temperature for 10 min. Each shrimp stomach
provided sufficient DNA extract for downstream analysis.

2.6.2. Library preparation and sequencing

The microbial 16 S rRNA V4 region was amplified using Q5 Hot Start High-Fidelity PCR master mix (NEB, Ipwich, USA) from the
extracted gDNA using the primer pair 515 F-806R (Walters et al., 2015) containing a partial Illumina Nextera adapter in their 5′ end.
The PCR condition used was 98 ◦ C - 30 s followed by 40–45 cycles of 98 ◦ C - 5 s, 55 ◦ C - 10 s and 72 ◦ C - 15 s. The PCR products were
cleaned using OmegaBiotek NGS total pure bead and subsequently used for index PCR reaction to incorporate dual-index barcode and
the remaining Illumina adapter. The index PCR products were pooled, bead-purified and quantified using Denovix high-sensitivity
fluorescence quantification kit (Denovix, Delaware, USA). The library was sequenced on an ISeq100 (Illumina, San Diego, CA)
using the run configuration of 1 × 300 bp as per the recommendation for 16 S V4 sequencing on this system, performed at GeneSEQ
Pte. Ltd. (Selangor, Malaysia).

2.7. Microbiota data and statistical analysis

Single-end demultiplexed fastq files generated were trimmed with cutadapt v1.18 to remove the non-biological forward and
reverse primer sequences located on the 5′ and 3′ ends of each read, respectively. The trimmed reads were used as the input for
amplicon sequence variant (ASV) generation and abundance table construction using dada2 (Callahan et al., 2016) which is part of the
QIIME2 v2020.8 pipeline (Bolyen et al., 2019). Taxonomic assignment of the ASV used the QIIME2 scikit-learn naive Bayes
machine-learning classifier (Bokulich et al., 2018) that was trained on the Greengenes 99% OTUs 16 S rRNA V4 gene sequences
(DeSantis et al., 2006). Non-mitochondrial and non-chloroplast ASV that were classified at least to the phylum level were used to
construct ASV abundance table. The filtered abundance table, taxonomic assignment output and sample metadata were analysed on
the MicrobiomeAnalyst webserver (Chong et al., 2020).
All other data in this experiment were presented as mean ± SE (n = 3) and subjected to one-way analysis of variance using the SPSS
statistical software (SPSS version 20.0; SPSS Inc., Chicago, IL, USA). Prior to analysis, data were subjected to homogeneity of variance
and if necessary were arcsine transformed. Differences among means were identified by Duncan’s post-hoc test, and effects with a
probability of P < 0.05 were considered statistically significant.

Table 2
Growth performance, feed utilization efficiency and survival of Penaeus vannamei fed experimental diets with increasing montmorillonite clay levelsa.
Montmorillonite clay (% of diet)

Parameters 0 0 0.25 0.50

(Negative control) (Positive control)

Initial weight (mg) 80 ± 2 70 ± 3 70 ± 4 80 ± 5

Final weight (mg) 170 ± 5 106 ± 1 160 ± 9 160 ± 9
Weight gainb (%) 122.8 ± 10.2 116.8 ± 8.2 115.0 ± 4.2 110.9 ± 5.5
Daily weight gainc (g/day) 6.63 ± 0.42 6.11 ± 0.24 6.15 ± 0.38 6.01 ± 0.37
SGR (%/day)d 5.71 ± 0.33 5.52 ± 0.28 5.47 ± 0.14 5.33 ± 0.19
FCRe 0.90 ± 0.04 1.03 ± 0.06 1.03 ± 0.03 1.07 ± 0.01
Feed intakef (g/shrimp/d) 3.91 ± 0.08 4.24 ± 0.12 4.28 ± 0.11 4.28 ± 0.12
Survivalg (%) 96.6 ± 1.6 95.0 ± 2.8 96.6 ± 3.3 95.0 ± 2.8
a
All values are mean ± S.E. of triplicate groups of shrimp. Different superscripts in the same row indicate significant differences at P < 0.05.
b
Weight gain (%) = [(final weight (mg) – initial weight (mg))/ initial weight (mg)] × 100
c
Daily weight gain (g/d) = (final weight (mg) – initial weight (mg))/ time (days)
d
Specific growth rate (%/d) = [(ln(final weight (mg)) – ln(initial weight (mg))/ time (days)] × 100
e
Feed conversion ratio (FCR) = total dry weight of feed offered/ total wet weight gained
f
Feed intake (g/shrimp/d) = (daily feed intake (g)/ average body weight (g)) × 100
g
Survival = (final fish number)/(initial fish number) × 100

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3. Results

3.1. Scanning electron micrographs and mineral composition

SEM micrograph of the montmorillonite clay, Calibrin®-Z, at 12,000x magnification showed the numerous stacked, latticed sur­
faces and interconnecting pores of the thermally-activated clay (Fig. 1A). Fig. 1B showed the presence of opal lepispheres which are
micro-spherical aggregates of silicon dioxide, interspersed among the clay lattices, which enhances thermal activation. Preliminary
EDX analysis for elemental chemical composition on selected surfaces of the clay samples showed C, O and Si as the major elements
together with the presence of Al, Mg, Br, K, Ca, Fe and Cu (data not shown).

3.2. Growth performance and feed utilization

All experimental diets were well received by the shrimp, and by the end of the feeding trial, shrimp more than doubled their initial
body weight (Table 2). The inclusion of dietary CL did not negatively influence growth, feed utilization efficiency, feed intake and
survival of shrimp, and all parameters were not significantly different (P > 0.05) (Table 2). Feed conversion ratio (FCR) was excellent
with values close to 1.0 and shrimp survival were 95% or higher in all treatment groups.

3.3. Disease challenge with VPAHPND

Shrimp in the negCON group (not challenged with VPAHPND) showed an average survival of 95.8 ± 2.1% over the seven days of the
disease challenge trial (Fig. 2). There was only two shrimp mortality out of a total of 48 shrimp in the negCON group. Shrimp in the
VPAHPND-challenged posCON group had the lowest average survival of 39.6 ± 10.4%, which was significantly lower (P < 0.05)
compared to the negative control. The infected shrimp showed clinical signs of the AHPND disease that included lethargy, a pale
atrophied hepatopancreas and empty gastrointestinal tract. The survival of shrimp fed CL-supplemented diets, irrespective of dietary
level, were not significantly different compared to the unchallenged negCON group (Fig. 2). There were no significant differences in
survival of shrimp fed either 0.25% or 0.5% CL during the disease challenge trial.

3.4. Bacteria counts in the hepatopancreas

The PVC and TCBC in the hepatopancreas of P. vannamei fed with the posCON diet after VPAHPND challenge were significantly the
highest (8.0 ± 0.2 log10 CFU g− 1 and 8.1 ± 0.1 log10 CFU g− 1, respectively) compared to the hepatopancreas of shrimp in other dietary
groups (Fig. 3). The negCON group served as an environmental control and showed the lowest bacterial counts compared to shrimp in
bacteria-challenged groups. Shrimp fed with CL supplemented diets, irrespective of dietary levels, showed significantly lower PVC and
TCBC counts compared to posCON shrimp (Fig. 3) and mostly not significantly different compared to the negCON shrimp.

3.5. Histopathology of hepatopancreas

The normal hepatopancreas of P. vannamei without being infected with AHPND (negCON group, Fig. 4A) composed of a series of
“star-like” tubules. Different epithelial cells such as B-cells and R-cells were present. In the VPAHPND-challenged groups, moribund

Fig. 2. Cumulative survival (n = 3) of P. vannamei fed the control diet (positive control) or montmorillonite clay-supplemented (Calibrin®-Z) diets
when challenged with V. parahaemolyticus (3HP strain). Shrimp in the negative control group was unchallenged. Different letters indicate significant
differences (P < 0.05).

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W.-K. Ng et al. Animal Feed Science and Technology 297 (2023) 115581

Fig. 3. Presumptive Vibrio spp. count (PVC) and total cultivable bacteria counts (TCBC) (mean ± SE, n = 3) in the hepatopancreas of P. vannamei
fed the control diet (positive control) or montmorillonite clay-supplemented (Calibrin®-Z) diets after challenged with V. parahaemolyticus (3HP
strain). Shrimp in the negative control group was unchallenged. Different letters indicate significant differences (P < 0.05) within the same group of
bacterial counts.

shrimp were confirmed to have AHPND by histological examination. Histopathology signs of infected shrimp (especially in the posCON
group, Fig. 4B) showed severe damage of hepatopancreas tubules, acute sloughing of epithelial cells and absence of B-, F-, and R-cells.
Hepatopancreas of shrimp fed diets with added 0.25% CL (Fig. 4C) or 0.5% CL (Fig. 4D) tended to show less damage compared to the
hepatopancreas of the posCON group.

3.6. Stomach microbiota

From a total of 309,885 filtered reads, 254 ASV were generated of which 93 ASV consist of more than or equal to two counts. The
average counts per sample was 25,823 (Min: 3857, Max: 47,000).

3.6.1. Alpha- and Beta-diversity of stomach microbiota

The Simpson index of the alpha-diversity plot measures the richness (number of ASV) and the evenness of ASV making up the
richness of the sample. A high Simpson index indicates high number of ASV and more similar proportions of ASV. When challenged
with VPAHPND, it was observed that the bacterial diversity in the posCON group was depressed compared to the unchallenged healthy
shrimp in the negCON group (Fig. 5). Supplementing shrimp diets with 0.25% CL tended to restore stomach bacterial richness and
diversity index much more than the 0.5% CL group (Fig. 5).
To compare the bacterial community structure among treatments, a beta diversity analysis, using the Bray-Curtis dissimilarity
metric, was performed. The principal component analysis plot exhibited the clustering of the bacterial population of the four treatment
groups (Fig. 6). Replicates in each treatment group showed separation by location in the plot with the exception of an outlier in the
0.5% CL replicate. ANOSIM analysis indicated a medium and significant dissimilarity among treatment groups (R=0.48148,
P < 0.004). AHPND infection clearly affected the configuration of the stomach microbiota as indicated by the tighter clustering of the
posCON group.

3.6.2. Stomach microbiota composition

At the phylum level, shrimp stomach bacterial community structure consisted mainly of Proteobacteria and Bacteroidetes with
Verrucomicrobia a distant third in relative abundance (Fig. 7). The accumulated abundance of these three phyla was higher than 90%
across treatment groups. The posCON group had increased relative abundance of Proteobacteria and decreased relative abundance of
Tenericutes compared to the negCON group. Increased relative abundance of Verrucomicrobia and Actinobacteria was observed in the
stomach of shrimp fed the CL-supplemented diets. At the 0.5% CL treatment group, slightly more abundance of Firmicutes was
observed.
A linear discriminant analysis effect size (LEfSe) analysis was conducted to identify specific bacteria genus that differentiate a
treatment group. This analysis was used to determine potential biomarkers in each stomach microbiome. Five bacteria of the genus
Demequina, Pseudoalteromonas, Tenacibaculum, Lysobacter and Marinimicrobium were the main contributors for the diversity differences
observed among treatment groups (P < 0.05) (Fig. 8). Higher abundance of Pseudoalteromonas, Tenacibaculum and Marinimicrobium
were found in the negCON group reflecting a broader taxonomical diversity in microbiota of healthy shrimp not infected with AHPND.
In shrimp fed the 0.25% clay diet, Demequina was identified as a potential biomarker and was significantly enriched in the stomach
compared to other treatment groups (Fig. 9). Despite alleviated abundance of Demequina in the stomach of shrimp fed the 0.5% CL diet,
the enrichment was not significantly different compared to the control treatments.

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(caption on next page)

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W.-K. Ng et al. Animal Feed Science and Technology 297 (2023) 115581

Fig. 4. Representative transverse sections of the hepatopancreas from P. vannamei fed the control diet (positive control) or montmorillonite clay-
supplemented (Calibrin®-Z) diets after challenged with V. parahaemolyticus (3HP strain). Shrimp in the negative control group was unchallenged.
(A) Hepatopancreas tissue of the unchallenged negative control shrimp showed normal appearance, which contained star-shaped lumen (L) and
normal developing B- and R-cells. (B) Hepatopancreas from the positive control group showed massive sloughing of hepatopancreatic tubule
epithelial cells and no B- and R-cells. (C) and (D) Hepatopancreas of shrimp fed the 0.25% or 0.5% clay diet, respectively, was observed to have less
damage and showed almost normal structure. Scale bars = 50 µm.

Fig. 5. Alpha-diversity plot (Simpson Index) of each sample from the negative control (negCON), positive control (posCON), 0.25% Calibrin®-Z
(0.25% CL) and 0.50% Calibrin®-Z (0.50% CL) experimental groups.

4. Discussion

In the present study, the pellet water stability of the experimental shrimp feed was similar to that of commercial feeds even after
four hours of immersion in seawater. Experimental shrimp feed added with 0.8% Pegabind® (pellet binder) imparted good water
stability. Shrimp being slow feeders with external mouth parts, good water stability of the experimental feeds was critical for optimal
feed intake and to ensure that the functional properties of the added MMT clay remained intact in water. The results of the growth trial
showed that MMT supplementation in the feeds of white shrimp did not significantly impact growth performance. The addition of
MMT in the form of the commercial product CL up to 0.5% of diet did not cause any toxicity or feed palatability issues as evident by the
high survival of shrimp and the excellent feed intake and FCR observed. Similarly, graded dietary MMT (99% purity) up to 1% did not
significantly influence growth, feed efficiency and survival of kuruma shrimp after five weeks (Zhang et al., 2022). The lack of impact
on growth was also reported in tilapia fed 0.15% MMT (Hu et al., 2007). In contrast, Shi et al. (2022) reported that turbot fed 0.3%
MMT showed significantly higher growth performance but not at dietary levels of 3% MMT. In a longer term 14-week feeding trial with
rainbow trout, Karimi et al. (2020) observed that fish fed 1, 2 or 4% MMT showed better growth performance, FCR and survival
compared to fish on a control diet without MMT supplementation. Apart from species specific differences, the discrepancies among
various published research on the effects of MMT on growth performance of aquatic animals are most likely due to the source and type
of MMT used. The purity, chemical composition and functional properties of natural MMT are very much dependent on the place
where the clay mineral was mined (Uddin, 2018). For example, in contrast to the results of the present study and that of Zhang et al.
(2022), Palm et al. (2015) reported that post-larvae of P. vannamei fed 2% MMT demonstrated a positive effect on growth, survival and
FCR. However, it should be noted that the MMT clay mineral used by Palm et al. (2015) contained only 40–60% actual MMT.
Comparisons of studies conducted with MMT should always consider the purity, composition and source of the MMT used. Very little
information is currently available on the impact of dietary MMT on growth performance of aquatic animals. Nevertheless, the use of
MMT as an ingredient in livestock feeds has generally reported beneficial results (Liu et al., 2021).
When challenged with VPAHPND, survival of shrimp fed 0.25% or 0.5% CL was high (83–94%) and was not significantly different
compared to the unchallenged negative control group (Fig. 2). Essentially, dietary MMT was able to protect shrimp from mortality
caused by bacterial toxins released by VPAHPND. To the best of our knowledge, this is the first report on the effectiveness of MMT as a

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W.-K. Ng et al. Animal Feed Science and Technology 297 (2023) 115581

Fig. 6. Principal component analysis (PCoA) plot based on Bray-Curtis dissimilarity matrix. Points in the plot represent individual samples ac­
cording to the treatment [negative control (negCON), positive control (posCON), 0.25% Calibrin®-Z (0.25% CL) and 0.50% Calibrin®-Z (0.50%
CL)]. Axis values indicates the percentage of variation in the input data that can be explained.

functional additive in the feeds of penaeid shrimp in mitigating the devastating effects of AHPND. We believe that the main mode of
action of MMT in mitigating AHPND is through the adsorption of the binary PirAB toxins on its surfaces and interlayers. Despite the
fact that there are no other reports on the ability of dietary MMT to bind bacterial toxins in aquatic animals, CL had been reported to be
able to bind Clostridium perfringens α-toxin and necrotic enteritis B-like toxin (NetB) in vitro (Lillehoj et al., 2016). These bacterial
exotoxins cause necrotic enteritis (NE), an intestinal infectious disease in poultry. Lillehoj et al. (2016) reported that NE-induced
broilers fed a diet supplemented with Ca-MMT plus a fermentable fiber and organic acid showed reduced gut lesions, improved
growth and increased serum antibody levels to α-toxin and NetB compared to broilers fed the basal diet. Supplementation with
Ca-MMT alone showed a trend toward reduced lesion scores and increased weight gain of broilers but it was not statistically significant
compared to controls. Bacterial toxins can migrate through interconnected pores and capillary channels (as shown in Fig. 1A) towards
internal binding sites. Various positively charged sites in the MMT structure such as the interlayer cations and edges of the octahedral
units provide the adsorption sites. Several of these cations on MMT surfaces were identified by EDX analysis in the present study. The
naturally occurring opal lepispheres (Fig. 1B) help maintain the layered sheet structures of CL and contributes to the high binding
capacity. Adsorptive binding forces on clay surfaces includes hydrophobic interactions, chelation, hydrogen bonding, electrostatic
attractions and van der Waals forces. In the present study, we believed that the adsorbed bacterial toxins by MMT were rendered
unavailable for absorption in the shrimp gut and eventually expelled from the body through fecal excretion. In addition, CL had been
shown, in vitro, to be a catalyst for disrupting quorum sensing in V. harveyi (Naik et al., 2018) which could then cause a significant
decrease in virulence expression of these pathogens. Dietary clay minerals and MMT had also been shown to improve non-specific
immune response which was credited for the enhanced disease resistance of shrimp to V. alginolyticus (Tan et al., 2014) and
rainbow trout to viral hemorrhagic septicemia virus infections (Karimi et al., 2020). There is still much room for research on the modes
of action of dietary clay products in the mitigation of diseases in aquatic animals.
The hepatopancreas of normal healthy P. vannamei had been reported to harbor bacteria, including a wide range of Vibrio spp.
(Gomez-Gil et al., 1998; Aranguren-Caro et al., 2020). During AHPND infection, bacterial and Vibrio spp. density in the hepatopancreas
of P. vannamei had been observed to increase concomitantly with disease progression (Soto-Rodriguez et al., 2015; Aranguren-Caro
et al., 2020). Similarly, in the present study, we observed significantly higher levels of TCBC and PVC in the hepatopancreas of the
AHPND-challenged postCON group (Fig. 3). In their review paper, Kumar et al. (2020) postulated that once VPAHPND colonizes the
stomach, it will release the PirAB toxin and also activates the Rho pathway. Activation of the Rho-signaling pathway will disrupt the
integrity of the tight junctions between the stomach epithelial cells producing intercellular gaps that will allow both the bacteria and

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Fig. 7. Mean relative abundance of bacterial phylum in different treatment groups [negative control (negCON), positive control (posCON), 0.25%
Calibrin®-Z (0.25% CL) and 0.50% Calibrin®-Z (0.50% CL)]. Each bacterial phylum is represented as a different color in the bar chart. The
combined proportion total to 1 for each treatment group.

Fig. 8. Linear discriminant (LDA) effect size (LEfSe) analysis to determine bacterial genera most likely to explain differences between treatments
[negative control (negCON), positive control (posCON), 0.25% Calibrin®-Z (0.25% CL) and 0.50% Calibrin®-Z (0.50%CL)]. Histogram shows the
LDA scores computed for bacterial genera among different treatment groups. No FDR correction was performed for this analysis but a lower raw p-
value cut-off of 0.05 was used due to the smaller sample size in each treatment group.

toxins to migrate to the hepatopancreas. Restrepo et al. (2021) observed that bacteria from the Order Vibrionales increased from
18.1% to 55.3% relative abundance in the hepatopancreas of white shrimp during AHPND infection. In the present study, the addition
of 0.25 or 0.5% CL in the feed significantly reduced the total bacterial and Vibrio counts found in the hepatopancreas (Fig. 3). This
seems to indicate that dietary MMT might have prevented the bacterial toxins from disrupting the integrity of the stomach lining
barrier. This line of reasoning will require further experimental evidence as a detailed understanding on the pathogenesis of AHPND is
still evolving. Nevertheless, Zhang et al. (2022) had shown that kuruma shrimp fed 0.05% MMT exhibited higher gut villus height and
width with up-regulated levels of intestinal mucosal barrier-related genes (fasciclin-II and integrin). The addition of MMT seems to have
improved the intestinal barrier function in shrimp. Similarly, Shi et al. (2022) observed significantly enhanced intestinal fold length
and wall thickness in turbot fed 0.3% MMT. Furthermore, histopathological examination of the hepatopancreas, the main target of the

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Fig. 9. Abundance of the bacterial genus Demequina identified to be one of the statistically and biologically informative features differentiating the
0.25% Calibrin®-Z (0.25% CL) treatment group (and possibly 0.5% CL treatment group) from non-clay treatment groups [negative control (neg­
CON), positive control (posCON)].

PirAB toxins, confirmed that shrimp fed MMT-added diets showed very little damage (Fig. 4 C, 4D) compared to AHPND-challenged
shrimp fed the control diet without any added MMT (Fig. 4B). This showed that little or no PirAB toxins migrated to the hepato­
pancreas to cause tissue damage as the adsorbed toxins by MMT were likely moved along the gut away from the hepatopancreas by
peristalsis and eventually excreted via fecal matter.
Stomach microbial diversity of infected shrimp was markedly reduced by AHPND compared to healthy shrimp (Fig. 5). Similarly,
Restrepo et al. (2021) reported that the diversity of microbiota in the stomach of AHPND-infected shrimp was significantly lower
compared to healthy shrimp. Furthermore, AHPND infection clearly affected the bacterial community structure of the shrimp stomach
and significant dissimilarity was observed among the treatment groups (Fig. 6). Similarly, using the Bray-Curtis dissimilarity metric,
Restrepo et al. (2021) reported that shrimp stomach microbiota clustered depending on the health status of shrimp. Zheng et al. (2016)
reported that the bacterial community in white shrimp was highly dependent on its health status. Not much information is currently
available on stomach microbiota changes in response to AHPND, but in general, it is known that a higher gut bacterial diversity reflects
better gut health and subsequent animal health. At the phylum level, we identified Proteobacteria and Bacteroidetes as the main
groups (Fig. 7) similar to that reported by Zheng et al. (2016). We observed that under AHPND infection, the relative abundance of
Proteobacteria was increased as compared to unchallenged shrimp. Similarly, Restrepo et al. (2021) reported an increase in relative
abundance of Proteobacteria from 89.8% in the stomach of healthy shrimp to 99.8% in AHPND-infected shrimp. We identified bacteria
of the genus Pseudoalteromonas, Tenacibaculum, and Marinimicrobium as members of the healthy microbiome and Lysobacter as being
relatively enriched in the stomach of AHPND-infected shrimp (Fig. 8). The actual roles played by these bacterial community changes
remains to be elucidated.
The present study is the first report on the modulation of shrimp stomach microbiota by dietary MMT during a disease infection.
The addition of MMT in the diet of white shrimp was observed to increase microbiota diversity, especially in the 0.25% CL group.
Similarly, Shi et al. (2022) reported that turbot fed 1% MMT significantly increased microbiota α-diversity in the gut. Shrimp fed
0.25% CL showed the highest relative abundance of Verrucomicrobia and Actinobacteria, followed by the 0.5% CL group. Firmicutes
relative abundance was more prevalent in the 0.5% CL group compared to other treatment groups. Changes in the relative abundance
of gut bacteria had also been reported for tilapia (Hu et al., 2007) and kuruma shrimp (Zhang et al., 2022) fed MMT. In the present
study, abundance of the bacterial genus Demequina was preferentially enriched in the stomach of MMT-fed shrimp, especially at the
0.25% level (Figs. 8 and 9). Demequina is a potential beneficial bacterium that had been reported to have amylase and xylanase
producing genes (Xin et al., 2009; Al-Naamani et al., 2015). Apart from the ability of MMT to bind PirAB toxin and mitigate
AHPND-inflicted stomach dysbiosis, the ability of dietary MMT in modulating stomach microbiota might also be due to the ability of
MMT to adsorb and immobilize bacteria selectively on their surfaces and interlayers (Liu et al., 2021) giving rise to different bacterial
profiles observed in this study.
In conclusion, dietary MMT can be used as an alternative to antibiotics for the mitigation and prevention of the AHPND pandemic in
shrimp aquaculture. It provides a natural solution to a present critical problem in shrimp farming sustainability. In the present study,
we intentionally used shrimp sizes that are known to be most vulnerable to AHPND in the production cycle. Based on the laboratory
results, we would suggest that CL at dietary levels of at least 0.25% be added in commercial shrimp feeds as an “insurance policy”
against production and economic losses from potential AHPND outbreaks in shrimp farms. Further longer-term and field-based studies

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W.-K. Ng et al. Animal Feed Science and Technology 297 (2023) 115581

are warranted to fully elucidate the beneficial impact of MMT in the feeds of farmed aquatic animals.

CRediT authorship contribution statement

Wing-Keong Ng: Conceptualization, Supervision, Methodology, Writing. Mei-Ling Mong: Data collection, Project administration,
Data analysis. Abdul-Azim Abdul-Hamid: Data collection, Data analysis.

Declaration of Competing Interest

The authors declare that there is no known conflict of interest. This research did not receive any specific research grant from
funding agencies. Subsequent publication cost of the manuscript was contributed by Amlan International Inc., USA.

Acknowledgments

Commercial names of products described in this paper do not denote endorsement by the authors. We would like to thank Dr.
Maximillian Sim (formerly of Amlan International Inc., USA) for providing the Calibrin®-Z utilized in this study. The efforts of Dr.
Victor Suresh (United Research Pte. Ltd., Singapore) in procuring the feed binder, Pegabind® from Bentoli AgriNutrition Co. Ltd.
(Thailand) is gratefully acknowledged. We thank Prof. Timothy Flegel (Centex Shrimp, Mahidol University, Thailand) for providing
the Vibrio parahaemolyticus (3HP strain) and Mr. Ung Eng Huan (BioValence Pte. Ltd., Malaysia) for the importation logistics.

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