Detection of ascaris suum in the livers of chickens infected naturally by the nested multiplex PCR assay

turally 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 
Detection of Ascaris suum in the Livers of chickens infected naturally by the nested multiplex PCR assay 
608 Vietnam Journal of Agricultural Sciences 
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. 
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, Germany) following the 
manufacturer's instructions. DNA concentration 
was measured by NanoDrop 2000 
(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). 
Nested multiplex PCR assay 
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 
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). 
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 
Macroscopic inspection on the liver surfaces 
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 
Ascarid specific universal 
Primers (First PCR) 
Ascarid 
Forward: 5’-CGTCGGTAGCGATGAAAGGT-3’ 
Reverse: 5’-TTAGTTTCTTTTCCTCCGCT-3’ 
Species-specific primers 
(second PCR) 
T. canis 
Forward: 5’-CTCGAGTCGACGAAGTATGTAC-3’ 
Reverse: 5’-AATTGGGCCGCCCATCATAC-3’ 
T. cati 
Forward: 5’-GTAAGATCGTGGCACGCGTACGTA-3’ 
Reverse: 5’-TCTTTGATGTCAAGACTTCAGCGC-3’ 
A. suum 
Forward: 5’-TCAACATTCATAGAGAATGGCATGT-3’ 
Reverse: 5’-TACATCATTATTGTCACGCTCATCT-3’ 
Nguyen Thị Hoang Yen et al. (2020) 
https://vjas.vnua.edu.vn/ 609 
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. 
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). 
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, 
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 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. 
Additionally, chickens were reared in old 
pigsties in some of the chicken farms (unpublished
Detection of Ascaris suum in the Livers of chickens infected naturally by the nested multiplex PCR assay 
610 Vietnam Journal of Agricultural Sciences 
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. 
Conclusions 
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 
direct proof of the infectivity of larvae to a new 
host. However, the results of this study 
provided the evidence of chickens ingesting A. 
suum eggs via exposure to contaminated soil. 
Thus, people need to made aware of the 
importance of treating pig manure before 
fertilizing plants, especially those with soil-
related occupations. This paper also provides 
critical information on the potential evidence of 
A. suum infections in humans in Vietnam. 
Further studies need to be conducted to evaluate 
A. suum infections in humans in this country. 
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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