Vô hoạt bào tử Microsporidian Enterocytozoon hepatopenaei (EHP): 1 phút ở 75°C

Aquaculture 533 (2021) 736178

Available online 30 November 2020

Microsporidian Enterocytozoon hepatopenaei (EHP) spores are inactivated in

1 min at 75 ◦C

Natthinee Munkongwongsiri

, Diva January Aldama-Cano

, Rungkarn Suebsing

Dararat Thaiue

, Tharinthon Prasartset

, Ornchuma Itsathitphaisarn

Kallaya Sritunyalucksana

Aquatic Animal Health Research Team (AQHT), Integrative Aquaculture Biotechnology Research Group, National Center for Genetic Engineering and Biotechnology

(BIOTEC), National Science and Technology Development Agency (NSTDA), Yothi Office, Rama VI Rd., Bangkok 10400, Thailand

Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Faculty of Science, Mahidol University, Rama VI Rd., Bangkok 10400, Thailand

Department of Biochemistry, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok 10400, Thailand

Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok 10400, Thailand

ARTICLE INFO

Keywords:

Enterocytozoon hepatopenaei (EHP)

Spore extrusion

Heat treatment

Infectivity test

Oral injection

Feeding

ABSTRACT

The microsporidian Enterocytozoon hepatopenaei (EHP) is a major threat for shrimp culture. We hypothesized that

commercial feeds processed at 75 ◦C or higher would present no risk for EHP transmission, even if they test

positive for EHP by PCR. To test this hypothesis, the purified EHP spores (1 ×10

spores) were incubated at

25 ◦C (non-heated) or 75 ◦C (heated) for 45–60 s prior to determining viability by polar tube extrusion assay.

Heat at 75 ◦C prevented extrusion when compared to spores incubated at 25 ◦C, confirming inactivation even

though PCR detection of EHP was still positive. Further tests were carried out using oral injection of heated and

non-heated spores. At day 14 post injection, SYBR Green-qPCR based on the EHP spore wall protein (SWP)

sequence revealed 1063–36,449 copies of EHP per ng DNA in the hepatopancreatic tissues of 3 from 4 shrimp

given non-heated spores while shrimp given spores heated at 75 ◦C for 45 s or 60 s showed no detectable copies.

In another experiment, shrimp were fed EHP-PCR-positive, commercial feed samples (CS1 and CS2) containing

the equivalent of 2.2 ×10

and 4.8 ×10

EHP copies/day for 21 days. The positive control consisted of EHP-

PCR-negative feed supplemented with active EHP spores (2.0 ×10

spores/day) fed for 14 days followed by

feeding with un-supplemented, EHP-PCR-negative feed from day 15 to 21. The negative control consisted of EHP-

PCR-negative, commercial feed. After 21 days, all shrimp in the positive control group showed EHP infection by

SYBR Green-qPCR and in situ hybridization. In contrast, shrimp fed with the negative control feed and the two

PCR-positive commercial feed samples (CS1 and CS2) showed no signs of EHP infection. The results supported

our hypothesis that CS1 and CS2 feeds contained inactive EHP DNA. Considering the heat exposure during

manufacture (equal to or exceeding 75 ◦C for 60 s), such feeds should be regarded as posing no risk for EHP

transmission.

1. Introduction

Hepatopancreatic microsporidiosis (HPM) is a newly emerging,

penaeid shrimp disease in the Australasian region. It is caused by

Enterocytozoon hepatopenaei (EHP) that was first reported from Thailand

in 2004 but not characterized until 2009 (Chayaburakul et al., 2004;

Tourtip et al., 2009). It is currently known to infect the giant tiger

shrimp Penaeus monodon and the white leg shrimp Penaeus vannamei,

and it is suspected to also infect Penaeus japonicus (Hudson et al., 2001).

High prevalence of EHP in cultivated stocks of P. vannamei in Thailand

was first discovered in a cohort study of shrimp ponds in 2015 (San-

guanrut et al., 2018), prompting publication at the NACA website

(www.enaca.org) of an urgent warning to the global shrimp aquaculture

industry the same year to beware of its spread (Sritunyalucksana et al.,

2015).

It is now known that EHP is highly prevalent in several countries in

* Corresponding author at: Aquatic Animal Health Research Team (AQHT), Integrative Aquaculture Biotechnology Research Group, National Center for Genetic

Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Yothi Office, Rama VI Rd., Bangkok 10400, Thailand

E-mail address: kallaya@biotec.or.th (K. Sritunyalucksana).

Contents lists available at ScienceDirect

Aquaculture

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

https://doi.org/10.1016/j.aquaculture.2020.736178

Received 8 June 2020; Received in revised form 1 November 2020; Accepted 17 November 2020

Aquaculture 533 (2021) 736178

Asia and that heavy infections are associated with retarded shrimp

growth and may be with white feces syndrome (WFS) where both can

lead to significant decreases in profits from harvested ponds (Flegel,

2020; Flegel, 2012; Flegel and Sritunyalucksana, 2018; Rajendran et al.,

2016; Thitamadee et al., 2016). Because of these potential losses, there is

growing attention on EHP control, with particular focus on the exclusion

of EHP from cultivation ponds. Important current strategies are to use

SPF broodstock shrimp that have EHP on their pathogen exclusion list.

Another is to cease the use of fresh and living broodstock feeds that may

be possible carriers of EHP. Because of attention on the possibility of

EHP in broodstock feeds, some hatchery and farm operators have begun

to test dry feeds and their ingredients for EHP by PCR and to reject

positive products for fear they may cause EHP infections. However, we

hypothesized that dry shrimp feeds that have gone through normal

manufacturing processes of mixing, pelleting and drying have reached

sufficient temperatures for a sufficient time to inactivate any EHP that

might have been present in feed ingredients. To test this hypothesis, we

performed experiments in which viable EHP spores (determined by

spore extrusion assay) were treated or not at 75 ◦C for 45 to 60 s, after

which they were used in viability assays and shrimp challenge tests to

determine the effect of heating on spore viability and infectivity.

2. Materials and methods

2.1. Preparation of EHP working spore batches

The effect of heat at 75 ◦C was tested on EHP activity by determi-

nation of the extrusion capability in vitro and infectivity via oral injection

and feeding. Shrimp with severe EHP infection were collected from

shrimp farms in Pathum Thani province, Thailand. Upon arrival at the

laboratory, the shrimp were anesthetized by immersion in ice water

before removal of the hepatopancreas (HP) (the target organ of EHP).

The EHP spores were purified from the homogenized HP following the

protocol described by Aldama-Cano et al. (2018) and kept in 1×PBS

(137 mM NaCl, 2.7 mM KCl, 10 mM Na

HPO

, 1.8 mM KH

, pH 7.4)

at room temperature (but for no longer than 4 days) as an initial stock

until use. The concentration of spores in each purified suspension was

determined by hemocytometer and adjusted to a working spore sus-

pension of either ~1 ×10

or ~ 2 ×10

spores/ml. The viability of each

purified spore batch was determined by spore polar tube extrusion assay

(Aldama-Cano et al., 2018). Briefly, the spore suspensions were incu-

bated with 2% Phloxine B solution for 10 min at room temperature

(25 ◦C) before observation by light microscopy in 3 replicates to count

the number of extruded spores in a total of 100 spores observed to yield a

mean % extrusion. Batches with extrusion less than 30% were not used.

2.2. Heat treatment of spore suspensions for extrusion assay

The heat treatments of EHP spores were carried out as four separate

experiments, each employing a different working spore suspension.

Based on equipment availability, two initial experiments were per-

formed using a water bath as the heat source while 2 later experiments

were done using a heating block as a heat source. The heat treatment

conditions were at 75 ◦C for 45 s or 60 s, in parallel with the non-heated

control treatment at 25 ◦C. For treatments, Eppendorf tubes (1.5 ml

containing 999

l of 1x PBS buffer) were placed in a heating source

accompanied by one tube containing buffer with a thermometer. When

the thermometer reached the desired temperature, a quantity of 1

from a working spore suspension (1 ×10

spores/ml) was added to each

tube to yield a final suspension of ~1 ×10

spores/ml. At the desired

exposure time, the tubes were removed and cooled to room temperature

before carrying out spore extrusion assays with Phloxine B to compare %

extrusion among the two heat treatments and the non-heated control.

The heat-treatment was done in continuous waves of 3 treatment and 1

temperature-control Eppendorf tubes at a time.

2.3. Detection of EHP by nested PCR and SYBR green-qPCR

The quality and quantity of DNA extracts were determined by

spectrophotometry prior to use as the DNA template for nested PCR

detection using the EHP-SWP nested PCR method (Jaroenlak et al.,

2016) or the SYBR green-qPCR method with SWP primers, the latter

developed in this study. The EHP-SWP nested PCR method was used to

detect EHP-DNA in the non-heated and heated spore preparations and in

shrimp orally injected with EHP spores. The SYBR green-qPCR was used

to check the copies of EHP/ng total DNA in shrimp following oral in-

jection, in the commercial feeds (CS1 and CS2) and in shrimp after

feeding with commercial feeds or EHP spore-supplemented feed.

For the EHP-SWP nested PCR method, the PCR mixture for both the

first step and the nested PCR step contained 1×OneTaq Hot Start Master

Mix (NEB) and 0.2

M of each primer. The templates used were either

25 ng of total DNA extracted from purified spores,100 ng of total DNA

extracted from shrimp hepatopancreatic tissue or 10

copies of positive

control SWP plasmid. For the second (nested) PCR step, the template

consisted of 1

l of the final reaction solution from the first PCR step. The

expected size for PCR products of the first step and nested PCR steps

were 514 and 148 bp, respectively. The amplicons were analyzed by

1.5% agarose gel electrophoresis with ethidium bromide staining and

using a 2 log DNA ladder marker (New England Biolabs, USA).

Determination of EHP copy numbers was carried out by SYBR green-

qPCR analysis in a 20

l reaction volume containing 1×SYBR green PCR

Master mix, 10 ng of total DNA extracted from shrimp hepatopancreatic

tissue, 0.2

M of SWP-1F (TTG CAG AGT GTT GTT AAG GGT TT) and

SWP-2R (GCT GTT TGT CTC CAA CTG TAT TTGA) primers. For con-

struction of the standard curve, the set of reactions was prepared to

contain a plasmid solution with different concentrations of the EHP-SWP

plasmid ranging from 0 to 10

(0, 100, 10

, 10

) copies/

l. The

qPCR profile was set at 95 ◦C for 15 s followed by 40 cycles of 95 ◦C for

15 s, 64 ◦C for 30 s and 72 ◦C for 30 s. This was followed by a dissociation

stage of 95 ◦C for 15 s, 60 ◦C for 1 min, 95 ◦C for 15 s and 60 ◦C for 1 min.

All reactions were run with two replicates. After the qPCR amplification,

the baseline and threshold were defined by using the ABI Prism 7500

Sequence detection software (AB Applied Biosystems Easter City, CA).

The standard curve generated is shown in Fig. 1.

Since SWP is a single-copy gene (Wiredu-Boakye et al., 2017), we

considered that the qPCR copy number (based on the standard curve

prepared using a plasmid containing the qPCR target sequence) was

approximately equivalent to the presence of an equal number of EHP

spore nuclei. To test this, relationship we prepared suspensions of spores

at 1.3 ×10

and 1.3 ×10

per

l as determined by hemocytometer (gold

standard) for DNA extraction using the phenol-chloroform method.

Then the extracted DNA was used as the template for qPCR, yielding the

result of 3.2 ×10

copies and 3.5 ×10

copies per

l, respectively. The

copy numbers for hemocytometer and qPCR were roughly comparable.

However, the DNA extracts from HP tissue include EHP nuclei in both

spores and developmental stages of plasmodia and may be dominated by

host DNA. Thus, it is possible that the dominance of host DNA might

have had some negative effect on the sensitivity of the qPCR. Thus,

although it is possible that the total EHP counts reported herein via qPCR

from tissue extracts might be somewhat lower than actual, it is of no

concern because we were only interested in determining whether

infection status of the tested shrimp was positive or negative.

2.4. Infectivity tests by oral injection

A total of 50 shrimp weighing approximately 3 g each were obtained

from the shrimp demonstration farm of the Chaipattana Foundation in

Chachoengsao province, Thailand. They were transported to the labo-

ratory in 100 L of well-aerated pond water (20 ◦C) and arrived at the

laboratory within 2 h. After arrival, they were transferred by net to a

laboratory holding tank (250 L) containing 100 L of aerated artificial

seawater (Marinium™) at 15 ppt (similar to the farm condition) and

N. Munkongwongsiri et al.

Aquaculture 533 (2021) 736178

continuously circulated through a filter pad at ambient temperature

(29–30 ◦C) for 2 days-acclimatization. The filter pad was removed,

cleaned and replaced every 3 days. The experimental groups were drawn

from this tank and transferred to smaller aquaria (100 L aquaria with 60

L artificial seawater) managed in the same way as the holding tank.

During acclimatization, 5 shrimp were arbitrarily selected and tested for

absence of EHP by nested PCR.

An EHP spore preparation with 77 ±2.7% extrusion was used for the

oral injection tests. There were three experimental groups (n =6 each).

One was injected with non-heated spores, one with spores heated at

75 ◦C for 45 s and one with spores heated at 75 ◦C for 60 s. A spore

suspension in 1

l (1 ×10

spores) from a working spore suspension of 1

×10

spores/mL was diluted to 50

l of PBS before injection directly

through the shrimp mouth into the stomach. After injection, shrimp

were reared in separate plastic tanks containing 30 L of 15 ppt saline

water (Marinium™) with constant aeration for 14 days before the

hepatopancreata for each group) were collected and used to detect the

EHP infection levels by nested PCR. SYBR green-qPCR analysis was also

carried out for a sub-sample of 4 specimens from each group.

2.5. Feed supplementation with untreated EHP-spores

From a working suspension of 2 ×10

spores/ml (described above) a

quantity of 1

lL (2 ×10

spores) was infused into each single feed pellet

that was then left to air-dry for 15 min at room temperature before being

used as positive control in shrimp feeding trials. The EHP spore-

supplemented feeds were prepared freshly in the morning of each

experimental day. The extrusion rate of the spore batches was checked

daily before use in preparing the feeds (Table 1) and not used for more

than three days or when the extrusion rate was less than 30%. The

reason was to avoid problems related to gradual, daily reduction in the

% extrusion that occurs for each batch after preparation (Aldama-Cano

et al., 2018). In total, 6 batches of EHP spores were prepared during the

experimental period.

2.6. Trials with experimental and commercial feeds PCR-positive for EHP

The number of 80 experimental juvenile shrimp (average weight of

1.7 g) were obtained from the same farm that provided shrimp for the

Fig. 1. Real time PCR detection of EHP spore wall gene using the SYBR green-qPCR method. (A) amplification plot and (B) standard curve for determination of the

EHP copy numbers. The equation for the linear regression line to calculate copy number (X) from Ct value (Y) is Y = − 3.484×+ 38.84. (For interpretation of the

references to colour in this figure legend, the reader is referred to the web version of this article.)

N. Munkongwongsiri et al.

Aquaculture 533 (2021) 736178

oral injection experiment. The transportation, acclimatization and

management protocols for the experimental shrimp were similar to

those used for the experimental shrimp for oral injection. They were

acclimatized for approximately 2 days during which time they were

tested (sample n =5 tested individually) for absence of EHP by nested

PCR. They were starved for one meal before they were divided into

subgroups for experiments. They were maintained on a basal diet con-

sisting of a commercial pelleted feed (shrimp feed pellet no. 2) and fed at

5% body weight for 2 meals daily (0.085 g/day).

These tests included 4 experimental groups (n =10 each). These

consisted of the positive control group (Group 1) given feed supple-

mented with 2.0 ×10

unheated EHP spores, the negative control group

(Group 2) given feed supplemented with buffer containing no EHP

spores and two groups (Groups 3 and 4) that consisted of shrimp given

different commercial feeds (CS1 and CS2, respectively) that had given

positive qPCR test results for EHP (Table 2). The shrimp were housed in

individual aquaria so ingestion of the feeds with active spores could be

confirmed.

Originally, the trial was designed to end at 14 days based on our

previous experience that infections can be confidently detected in EHP

challenge tests within that timeframe (Salachan et al., 2017). However,

sub-sampling of 3 shrimp from each group on day 14 gave no EHP-

positive specimens in Groups 3 and 4, so we decided to extend the

trial another 7 days to allow more opportunity for infection with the

commercial feeds.

Each day for the first 14 days, shrimp in the EHP-supplemented feed

Group 1 and in the negative control, buffer-supplemented Group 2 were

fed one supplemented pellet as the first pellet in the first meal in the

morning of each day. After ensuring that each shrimp had eaten the

supplemented pellet, they were each given an additional 4 pellets from

the basal diet. For the second meal they were given 5 basal feed pellets.

Thus, the total spore dose received was 2.0 ×10

spores/day for 14 days

after which they were continuously fed with the basal, un-supplemented

feed from days 15 to 21 when the experiment ended.

The commercial feed pellets (CS1 and CS2) used to feed shrimp in

Groups 3 and 4 were originally designed for large shrimp and only 4

pellets per day were sufficient to reach the target of 5% body weight for

2 meals daily (i.e., 0.085 g/day). Thus, the 2 pellets per meal for each

shrimp were cut down to an appropriate size for the target shrimp before

feeding. Based on the qPCR results for pellets of CS1 and CS2, each

shrimp received the equivalent of 2.2 ×10

and 4.8 ×10

EHP copies/

day in 4 pellets, respectively, with continuous feeding for 21 days.

Table 3 summarizes the number of EHP copies that shrimp in the CS1

and CS2 received per day and the number of EHP spores that shrimp in

the positive control group received per day. On day 21, the shrimp were

processed for removal of their hepatopancreata and DNA was extracted

from each for individual quantification of EHP by SYBR green-PCR.

2.7. Statistical analysis

Results were compared by one-way ANOVA using SigmaStat

(Version 12.0) and differences were considered significant at p ≤0.05.

2.8. Histological analysis and in situ hybridization

For histological examination, the hepatopancreas from each experi-

mental shrimp was fixed with Davidson’s fixative for 24 h and processed

for routine histological analysis of H&E stained tissue sections (Bell and

Lightner, 1988). An adjacent section from the same paraffin block of

each individual sample was sectioned and processed for in situ hybrid-

ization assay. The primers ENF779 (5′- CAG CAG GCG CGA AAA TTG

TCCA-3′) and ENR779 (5′- AAG AGA TAT TGT ATT GCG CTT GCTG-3′)

were used with a plasmid template containing an EHP-SSU-rRNA gene

fragment to prepare a DIG-labeled SSU probe following the protocol

described by Tangprasittipap et al. (2013). The SSU probe was used for

in situ hybridization because it gave higher sensitivity than the SWP

probe by this technique.

3. Results and discussion

3.1. Heating at 75 ◦C prevents EHP spore extrusion

Results for the experiment on the effect of heat on spore extrusion are

shown in Table 4. For the first and second heating block trials at 75 ◦C

for 45 s and 75 ◦C for 60 s, no spore extrusion was seen while extrusion

for the non-heated spores was 61 ±2.0% and 77 ±2.7%, respectively.

For water bath heating, a very low extrusion of 0.33 ±0.58% was

observed for the treatment at 75 ◦C for 45 s, while no extrusion was seen

after heating at 75 ◦C for 60 s. The very low extrusion rate of 0.33% at

Table 1

The percentage extrusion of the purified EHP spores infused into shrimp feed pellets.

Spore preparation Batch #1 Batch #2 Batch #3 Batch #4 Batch #5 Batch #6

Feeding day 1 2 3 4 5 6 7 8 9 10 11 12 13 14

% extrusion 86 61 30 88 42 40 60 48 56 36 54 44 80 55

Table 2

EHP copy numbers in three commercial feed samples as determined by SYBR

green-qPCR. UD =undetectable.

Group Treatment (n =10) EHP copy

numbers/g feed

1 Commercial feed no. CS1 2.6 ±0.3 ×10

2 Commercial feed no. CS2 5.6 ±0.2 ×10

3 Commercial feed used to prepare positive and

negative control feed (basal feed)

Table 3

Feeding schedule for the EHP spore-supplemented feed (positive control) and

the EHP-PCR positive feeds (CS1 and CS2).

Feeding day EHP copy number in shrimp fed with

CS1* CS2* Positive control**

1 2.2 ×10

4.8 ×10

2.0 ×10

2 2.2 ×10

4.8 ×10

2.0 ×10

3 2.2 ×10

4.8 ×10

2.0 ×10

4 2.2 ×10

4.8 ×10

2.0 ×10

5 2.2 ×10

4.8 ×10

2.0 ×10

6 2.2 ×10

4.8 ×10

2.0 ×10

7 2.2 ×10

4.8 ×10

2.0 ×10

8 2.2 ×10

4.8 ×10

2.0 ×10

9 2.2 ×10

4.8 ×10

2.0 ×10

10 2.2 ×10

4.8 ×10

2.0 ×10

11 2.2 ×10

4.8 ×10

2.0 ×10

12 2.2 ×10

4.8 ×10

2.0 ×10

13 2.2 ×10

4.8 ×10

2.0 ×10

14 2.2 ×10

4.8 ×10

–

15 2.2 ×10

4.8 ×10

–

16 2.2 ×10

4.8 ×10

–

17 2.2 ×10

4.8 ×10

–

18 2.2 ×10

4.8 ×10

–

19 2.2 ×10

4.8 ×10

–

20 2.2 ×10

4.8 ×10

–

21 2.2 ×10

4.8 ×10

–

Totals 4.6 ×10

1.0 ×10

2.6 ×10

The number of EHP in CS1 and CS2 were determined by qPCR.

The number of EHP in the positive control group was determined by the

number of spores added to the feed.

N. Munkongwongsiri et al.

Aquaculture 533 (2021) 736178

45 s represented 1 in 100 spores in one of 3 replicates in one trial with

water bath heating. We believe this extrusion occurred during the

preparation steps before heat treatment. In contrast, extrusion for the

non-heated spores was high at 71 ±1.2%, and 79 ±1.53%, respectively.

3.2. Heated EHP spores are not infectious by oral injection

In the experiments on infectivity of heat-treated EHP spores by oral

injection, 2 shrimp in non-heated spore injection died after molting at

night and being partially eaten (appendages and eyes) by the other

shrimp in the group. Three out of four of the remaining shrimp injected

with non-heated spores gave PCR positive results for EHP infection. In

contrast, the two groups injected with spores heated at 75 ◦C for 45 s or

60 s (n =6 +6 =12) all gave negative PCR results for EHP-SWP nested

PCR (agarose gels not shown). In the sub-sample of 4 shrimp taken from

each of these groups for qPCR testing (Table 5), those from the group

injected with non-heated spores gave one specimen with undetectable

(UD) EHP and 3 specimens with 6489 ±351, 1093 ±63, and 36,449 ±

1266 copies of EHP/ng DNA. In all samples (4 +4 =8) from the other

groups fed heat-treated spores, EHP was undetectable (UD). These re-

sults indicated that the heated spore preparations were non-viable (i.e.,

unable to cause EHP infections). These results concurred with those

from the in vitro extrusion assay that gave negative extrusion results only

with the heated spores.

Similar effects of temperature on spore activity have been reported

for other microsporidians such as Encephalitozoon intestinalis, Enc. hellem

and Enc. cuniculi. The infectivity of microsporidian spores treated at

70 ◦C, were dramatically reduced to 84.4, 98.9, and 98.9% for Enc.

intestinalis, Enc. hellem and Enc. cuniculi, respectively (Li and Fayer,

2006). The fish microsporidian, Spraguea sp., demonstrated low polar

filament extrusion (3%) after its spores were exposed to 60 ◦C for more

than 5 min and almost 100% inactivation when spores were kept at this

temperature for 15 min (Leiro et al., 2012). In our study, the EHP spores

were found to be inactivated within a short time (1 min) at 75 ◦C. It is

important to emphasize that the heat exposure time count was not

started until the spore suspension had reached 75 ◦C. Thus, heat treat-

ment of large batches of feed or feed ingredients would need to be

adjusted to ensure that core heating temperature would reach 75 ◦C for

at least 1 min. Thus, the total heating time would vary depending sub-

strate quantity and form.

3.3. Heated, non-viable spores were PCR positive for EHP, as expected

When heated and non-heated spore preparations were subjected to

DNA extraction and purification followed by nested SWP-PCR, all gave

strong, positive EHP-PCR amplicons of 148 and 514 bp (Fig. 2). In the

samples with high copy numbers of the target, an additional band at

about 180 bp was also detected arising from interaction between re-

sidual primers in the template DNA from the first step PCR with those in

the second-step, nested PCR (Jaroenlak et al., 2016). The results

confirmed that PCR detection alone could not be used to determine EHP

spore viability and, thus, should not be used alone to screen shrimp feeds

or feed ingredients to assess whether they pose any risk for EHP trans-

mission. This is to be expected since DNA is known to be heat stable and

capable of remaining intact after heat treatment.

3.4. EHP-PCR-positive commercial shrimp feed is not infectious

In this experiment, 2 commercial feeds (CS1 and CS1) that had

previously tested positive for EHP by PCR were subjected to qPCR

analysis to determine the copy number of EHP per gram feed. The results

indicated the equivalent of 2.6 ±0.3 ×10

and 5.6 ±0.2 ×10

EHP

copies/g feed for CS1 and CS2, respectively (Table 2). These feeds were

given to test Groups 3 and 4, respectively, while the positive control

Group1 was given feed supplemented with active EHP spores and the

negative control groups was given feed supplemented with buffer

solution.

On day 14, 3 shrimp samples were taken from each of the 4 experi-

mental groups to test for the presence of EHP in the shrimp HP by PCR

analysis. All 3 sample from positive-control Group1 given feed supple-

mented with active EHP spores gave positive PCR results for EHP

(Fig. 3). In contrast 3 shrimp each from the negative control Group 2 and

the two commercial feed Groups 3 and 4 (positive for EHP by PCR) also

gave negative PCR results for EHP (Fig. 3). Because of this, it was

decided to extend the feeding a further 7 days to allow more opportunity

for infection from the commercial feeds.

During the extended time from day 15–21 (7 shrimp in each exper-

imental group), two shrimp (pre-molt, soft shells) died in Group 3 fed

with the commercial feed CS1. Thus, after the experiment ended on day

21, the remaining 5 shrimp from Group 3 and 5 arbitrarily selected

shrimp from the 7 shrimp remaining in the other 3 groups were tested

for EHP by PCR. All 5 shrimp from the positive control Group1 gave

Table 4

Determination of spore extrusion rate (%) after heat treatment. Tests were done

in 3 replicates (Rep) to obtain an average extrusion rate ±standard deviation

(SD).

Trial Spore extrusion rate (%)

Treatment Rep 1 Rep 2 Rep 3 Mean ±SD

1st with dry bath RT 61 59 63 61 ±2.0

75 ◦C / 45 s 0 0 0 0

75 ◦C / 60 s 0 0 0 0

2nd with dry bath RT 80 75 76 77 ±2.7

75 ◦C / 45 s 0 0 0 0

75 ◦C / 60 s 0 0 0 0

1st with water bath RT 70 72 72 71 ±1.2

75 ◦C / 45 s 0 0 0 0

75 ◦C / 60 s 0 0 0 0

2nd with water bath RT 81 79 78 79 ±1.5

75 ◦C / 45 s 0 1* 0 0.3 ±0.8

75 ◦C / 60 s 0 0 0 0

We believe this spore may have extruded prior to heat treatment.

Table 5

The quantity of EHP (copy number per ng DNA) in shrimp orally injected with

heated and non-heated EHP spores. UD =undetectable.

Experimental group Shrimp

no.

EHP copy number/ng DNA

Mean ±SD

Injected with EHP spores heated at

75 ◦C for 45 s

1 UD

2 UD

3 UD

4 UD

Injected with EHP spores heated at

75 ◦C for 60 s

1 UD

2 UD

3 UD

4 UD

Injected with non-heated EHP spores 1 6489 ±351

2 UD

3 1093 ±63

4 36,449 ±1266

Fig. 2. Agarose gel electrophoresis showing positive results for expected SWP-

PCR amplicons in both heat-treated and untreated EHP spore preparations. M:

Molecular marker (2 log DNA ladder marker), N: Negative Control, Lane 1:

Non-heated spores, Lane 2: Spores heated at 75

◦C/45 s, Lane 3: Spores heated

at 75 ◦C/60 s, P: Positive control.

N. Munkongwongsiri et al.

Microsporidian Enterocytozoon hepatopenaei (EHP) spores are inactivated in 1 min at 75 ◦C

Bài viết nghiên cứu chứng minh xử lý nhiệt 75°C/1 phút bất hoạt bào tử EHP gây bệnh trên tôm, thức ăn công nghiệp đạt nhiệt độ này an toàn, dù PCR dương tính.

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