Detection of Ascaris suum in the livers of chickens infected naturally by the nested multiplex PCR assay
ISSN 2588-1299
VJAS 2020; 3(2): 606-611
Vietnam Journal
of Agricultural
Sciences
Detection of Ascaris suum in the Livers of
Chickens Infected Naturally by the Nested
Multiplex PCR Assay
Nguyen Thi Hoang Yen1, Nguyen Thi Hop2, Tran Hai Thanh1,
Nguyen Van Phuong1, Nguyen Thi Hong Chien1, Bui Khanh
Linh1 & Do Trung Dung2
1Faculty of Veterinary Medicine, Vietnam National University of Agriculture, Hanoi
131000, Vietnam
2Department of Parasitology, National Institute of Malariology Parasitology and
Entomology, Hanoi 100000, Vietnam
Abstract
This study was conducted to detect Ascaris suum, Toxocara canis,
and Toxocara cati in naturally infected chicken livers, and then to
provide information related to ascarid infections in humans. Ninety-
four chicken liver samples collected at a fresh market were used for
this study. DNA was extracted from each minced liver sample (500
mg) by the alkaline lysis method using NaOH 50mM and Tris-HCl
(pH 8.0). The nested multiplex PCR assay using ascarid universal
primers was applied to amplify the ITS1 ribosomal RNA gene of A.
suum, Toxocara canis, and Toxocara cati in all the liver samples.
Then, species specific-primers were used to discriminate between A.
suum and Toxocara spp. The results showed that 2 of the 94 chicken
liver samples detected positive for the presence of A. suum DNA.
This study provided useful information and evidence about A. suum
infection in humans via exposure to contaminated soil or eating
raw/undercooked chicken livers in Vietnam.
Keywords
Ascaris suum, chicken liver, nested multiplex PCR, white spot
lesions
Introduction
Acaris suum, along with Toxocara canis and Toxocara cati, are
common roundworms in pig, dog, and cat intestines, and are
considered as the causal agents of ascarid larva migrans syndrome
(ascarid LMS) in humans. These roundworms have a direct life cycle
in which the process of egg embryonation occurs outside in the
environment, and they do not need any intermediate hosts to be at an
infective stage. Once definitive hosts ingest ascarid embryonated
eggs, larvae emerge in the digestive system, penetrate the intestinal
wall, and then migrate to the liver via the mesenterial blood veins.
Received: May 26, 2019
Accepted: July 12, 2020
Correspondence to
ORCID
Nguyen Thi Hoang Yen
0945
606
Vietnam Journal of Agricultural Sciences
Nguyen Thị Hoang Yen et al. (2020)
Chicken are one of the paratenic hosts of the
ascarid roundworm because this roundworm
does not develop into adult worms in chickens
(Taira et al., 2003; Azizi et al., 2007; Yoshihara
et al., 2008). Laboratory experiments of A. suum
infection in chickens have shown the pattern of
ascarid larvae distribution. After hatching in the
digestive system, ascarid larvae penetrated the
intestinal wall, migrated to the liver, lungs, and
eventually back to the liver (for T. canis) where
they remained in the carcass (for T. cati) (Okoshi
& Ushui, 1968; Taira et al., 2003) or were
eliminated from the chicken after 14 days
(Yoshihara et al., 2008). However, in natural
infections, chickens that are reared freely outside
in the environment (free-range chickens) could
have a higher risk of getting ascarid eggs from
the soil based on their temperament and become
a paratenic host for them, especially if pig
manure is used for fertilizer (Vu et al., 2007).
Because of this temperament, soil contaminated
with ascarid eggs could be evaluated in addition
to the detection of anti-ascarid antibodies in
chickens (Campos de silva et al., 2015). Thus,
the objectives of this study were to evaluate of
the contamination of chicken livers with A. suum,
T. canis, and T. cati and then provide evidence of
ascarid roundworm infections in humans through
contact with contaminated soil or eating
raw/undercooked chickens in Vietnam.
From the liver, larvae are carried to the lungs
using the efferent bloodstream, penetrate the
lung alveoli, and then migrate up the respiratory
tree. Finally, larvae are coughed up and
swallowed by the host to reach the small intestine
where they develop into adult worms. The
prepatent period is about 6-8 weeks.
Toxocariasis caused by T. canis and T. cati
was first recorded in the 1950s, followed by A.
suum infection, and impacts many countries
around the world (Beaver et al., 1952; Thompson
et al., 1986; Chomel et al., 1993; Magnaval et al.,
1993; Maruyama et al., 1996; Sakakibara et al.,
2002; Hoenigl et al., 2010; Izumikawa et al.,
2011; Penelli et al., 2011; Miller et al., 2015). In
Vietnam, some researchers have reported
toxocariasis in humans (Tran et al., 2014; Do et
al., 2016), however, there has been no
information about A. suum infection in humans.
The migration of ascarid larvae through the
human body can cause severe health problems
such as eosinophilia, fever, coughing, enlarged
liver, or pneumonia (Sakakibara et al., 2002;
Izumikawa et al., 2011; Lamberton & Jourdan,
2015). In some cases, larvae can migrate to the
brain or spinal cord causing neuro larva migrans
(NLM) (Inatomi et al., 1999; Umehara et al.,
2006) or eyes (Glickman et al., 1987). It is called
ascarid LMS in humans.
The transmission route of this infection
varies in different countries, including either by
accidental ingestion of egg embryonates in the
soil or contaminated vegetables/drinking water
(Matsuyama et al., 1998; Tokojima et al., 2004;
Izumikawa et al., 2011); or eating raw/undercook
meat and offals contaminated with the larval
parasites (Choi et al., 2012). The former methods
are considered the main routes in American or
Western European countries. By contrast, in East
Asian countries such as Japan and South Korea,
the later route has recently become more
important due to eating habits. In Vietnam, the
former routes could be considered as the main
methods due to the habit of using pig manure for
soil fertilizer in some locations (Vu et al., 2007).
However, the latter route has also become a risk
because of the culture exchange amongst Asian
countries, especially cuisine culture.
Materials and Methods
Parasites
A. suum, Toxocara canis, and T. cati adult
worms were collected from naturally infected
pigs, dogs, and cats, respectively, at local
abattoirs around Hanoi. They were identified by
morphology before performing the next steps.
The roundworms were then thoroughly washed
in saline solution and male worms were separated
and kept in 70% alcohol for DNA extraction.
Liver samples
Ninety-four chickens were purchased from a
fresh market in Trau Quy Town, Gia Lam
district, Hanoi, Vietnam in order to collect the
livers from naturally infected chickens. These
free-range chickens were reared freely and
derived from Hoa Binh, Bac Giang, and Hai
607
Detection of Ascaris suum in the Livers of chickens infected naturally by the nested multiplex PCR assay
Duong provinces. The chickens then were
necropsied to inspect the macropathology (white
spot lesions) on the liver surfaces. Next, the
livers were collected for the detection of A. suum
by the nested multiplex PCR assay.
following PCR mixture: 12.5μL Master mix 2X
(Phusa Biochem, Vietnam), 0.75μL of each
primer at concentrations of 0.3mM (Phusa
Biochem, Vietnam) and 10μL distilled water.
The thermal cycling profile was 94°C for 2min,
followed by 40 cycles with denaturation at 94°C
for 15s, primer annealing at 52°C for 30s, and
extending at 68°C for 1min, and a final
elongation step at 72oC for 7min. The first PCR
products were used as a template in the species-
specific nested multiplex PCR, in which multiple
primer sets were applied in the same tube with
0.75μL of each of species-specific forward and
reverse at concentrations 0.3mM. The PCR
conditions were similar to the first reaction, except
for primer annealing was at 57oC (Table 1) (Wang
et al., 2018).
DNA extraction
The genomic DNA was extracted from adult
A. suum, T. canis, and T. cati worms using a
Blood- Animal- Plant preparation Kit (Jena
Bioscience,
manufacturer's instructions. DNA concentration
was measured by NanoDrop 2000
Germany)
following
the
(ThermoFisher Scientific, US) to detect the
presence of DNA.
The alkaline-lysis method was employed for
extracting gDNA from the liver tissue. Five
hundred milligrams (500mg) of chicken liver
was homogenized in a 15mL tube provided by
BioMasher (Nippi, Tokyo, Japan), and 1.8 mL of
50mM NaOH was added. After boiling for 30
min, the samples were neutralized by 200μL of
1M Tris-HCl (pH 8.0). The mixture was vortexed
thoroughly and centrifuged at 14,000×g for
10min. The supernatant was transferred to a new
tube and stored at -20°C until analysis (Nguyen
et al., 2016).
The amplification products were analyzed
using 1.0% agarose gel electrophoresis. The
amplification bands were visualized under UV
light. A positive reaction was observed when the
appearance of the specific sized bands of 208bp,
325bp, and 233bp corresponding with T. canis,
T. cati, and A. suum, respectively (Wang et al.,
2018).
Results and Discussion
Nested multiplex PCR assay
Macroscopic inspection on the liver surfaces
The nested multiplex PCR assay was applied
to amplify a region of the ITS1 ribosomal RNA
gene of A. suum, T. canis, and T. cati. The PCR
reaction was performed in the total volume of 25
μl containing 1μL of template DNA and the
Most of the collected livers were normal in
color and structure. However, 15 of the 94
chickens showed macropathological damage on
their livers. White spot lesions ranging from
Table 1. Primer sequences that were used for the nested PCR and nested multiplex PCR (Wang et al., 2018)
Primers
Sequences
Forward: 5’-CGTCGGTAGCGATGAAAGGT-3’
Reverse: 5’-TTAGTTTCTTTTCCTCCGCT-3’
Ascarid specific universal
Primers (First PCR)
Ascarid
Forward: 5’-CTCGAGTCGACGAAGTATGTAC-3’
Reverse: 5’-AATTGGGCCGCCCATCATAC-3’
T. canis
T. cati
Species-specific primers
(second PCR)
Forward: 5’-GTAAGATCGTGGCACGCGTACGTA-3’
Reverse: 5’-TCTTTGATGTCAAGACTTCAGCGC-3’
Forward: 5’-TCAACATTCATAGAGAATGGCATGT-3’
Reverse: 5’-TACATCATTATTGTCACGCTCATCT-3’
A. suum
Vietnam Journal of Agricultural Sciences
608
Nguyen Thị Hoang Yen et al. (2020)
Figure 1. The white spot lesions on chicken livers infected naturally
1-10 in number and 0.1-0.5mm in diameter were
observed on both surfaces of the livers (Figure 1).
In addition, several different macropathological
features were observed on the liver surfaces, such
as damage caused by Marek’s disease. However,
those samples were excluded from this study.
positive control samples (ascarid adult-derived
DNA) were successfully amplified. Of the 94
livers collected, two of them were positive and
both of them showed amplification of a 233bp
band, which is specific for A. suum (Figure 2).
Meanwhile, Toxocara spp. DNA were not
detected in this study.
The observation of white spot lesions on the
liver surfaces was one piece of critical evidence
of the migration of ascarid larvae through the
chicken liver (Taira et al., 2003; Azizi et al.,
2007; Yoshihara et al., 2008). However, the
white spot lesions were only recognized on day 7
in chickens with double or triple A. suum
infections (Yoshihara et al., 2008). White spot
lesions were not observed if the chickens were
not reinfected or in the cases of early infection
(Yoshihara et al., 2008).
The presence and existence of larvae in the
liver depends on each agent. In the cases of T.
cati and A. suum, the larvae exist in the liver
around 14 days after the last experimental
infection (Azizi et al., 2007; Yoshihara et al.,
2008). For T. canis, after the larvae migrate to the
lungs, they migrate back to the liver and exist
there for ninety days (Okoshi & Usui, 1968;
Taira et al., 2003). The results of this study
showed that the two positive samples of A. suum
infection were not in livers showing white spot
lesions. Thus, the detection of A. suum herein
illustrated new infections of A. suum in those
naturally infected chickens. Investigation of
several free-range chicken farms showed that the
farmers often plant fruit trees such as pomelo
trees or litchi trees around and inside chicken-
rearing areas in order to provide shade for the
chickens, and they sometimes used pig manure to
fertilize these trees without treatment.
In natural infection, chickens can ingest eggs
more frequently, leading to a higher chance of
larvae presenting in liver. Moreover, raw liver is
also a special cuisine in some countries such as
Japan and South Korea. This is one of the
transmission routes by which humans get ascarid
larva migrans syndrome (ascarid LMS) (Choi et
al., 2012). Thus, the liver samples were the target
of selection for this study.
Detection of A. suum in the chicken livers
Before applying the nested multiplex PCR
assay for the naturally infected liver samples,
Additionally, chickens were reared in old
pigsties in some of the chicken farms (unpublished
609
Detection of Ascaris suum in the Livers of chickens infected naturally by the nested multiplex PCR assay
Note: Lane M: 100 bp DNA ladder marker; Lanes 1-3: positive controls (lane 1: T. canis DNA; lane 2: T. cati DNA; lane 3: A. suum
DNA); Lane 4: negative control; Lanes 5-6: A. suum DNA liver samples.
Figure 2. The results of Ascaris suum detection in naturally infected chickens
results from another study). Meanwhile, dogs or
cats were usually raised outside with no/little
chance of invading the chicken-rearing areas.
Thus, it is possible that the chance of ingesting A.
suum eggs was higher than with Toxocara eggs
in free-range chickens. Moreover, T.
malaysiensis was reported in cats in Hanoi (Le et
al., 2016). Thus, further studies need to be
conducted to clarify this issue.
Acknowledgements
This research was supported by grants from
ARES-CCD (Académie de Recherche et d’
Enseignement supérieur Wallonie-Bruxelles-
Commission
de
la
Coopération
au
Développement) in cooperation with Vietnam
National University of Agriculture.
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The detection of A. suum DNA in naturally
infected chicken livers is a direct proof of soil
contamination with A. suum eggs, but not a
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Further studies need to be conducted to evaluate
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