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To cite this article: Panarat Phadee , Osamu Kurata , Kishio Hatai , Ikuo Hirono & Takashi Aoki (2004) Detection and
Identification of Fish-Pathogenic Aphanomyces piscicida Using Polymerase Chain Reaction (PCR) with Species-Specific Primers,
Journal of Aquatic Animal Health, 16:4, 220-230, DOI: 10.1577/H03-047.1
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Journal of Aquatic Animal Health 16:220–230, 2004
q Copyright by the American Fisheries Society 2004
Aphanomyces piscicida (also known as A. in- omyces has been based on phenotype and char-
vadans; David and Kirk 1997) is the causative acteristics such as sexual versus asexual repro-
agent of mycotic granulomatosis (MG) in warm- duction (Scott 1961). However, the isolation and
water fish from Japan (Egusa and Masuda 1971; identification of pathogenic Aphanomyces spp. is
Miyasaki and Egusa 1972, 1973a, 1973b, 1973c; sometimes difficult (Willoughby and Roberts
Hatai 1980; Kurata et al. 2000). Aphanomyces pis- 1994) and time consuming (Einsele et al. 1997;
cicida is responsible for a number of fungus-as- Bretagne et al. 1998; Zhao et al. 2001) because
sociated diseases, including epizootic ulcerative the pathogen is poorly characterized and identifi-
syndrome (EUS) in freshwater and brackish-water cation requires a high level of technical expertise.
fish in the Asia–Pacific region (Miles et al. 2001), Pathogenicity to fish has been listed as one of the
red spot disease (RSD) in Australia (McKenzie and distinguishing features of A. piscicida (A. inva-
Hall 1976; Callinan et al. 1989), and ulcerative dans); several variety of other species of Aphan-
mycosis (UM) in the USA (Dykstra et al. 1989). omyces are incapable of sustained growth on fish
Infected fish usually present dermal ulcers and an tissues (Blazer et al. 2002). However, this is not
associated loss of scales, hemorrhage, edema, and a standard criterion for identification.
necrotic open ulcers on the body surface. It is usu- Recently, polymerase chain reaction (PCR)
ally found with aseptate hyphae extending to the methods have been used for the detection and iden-
skeletal muscle within granulomas (Callinan et al. tification of fungal diseases in humans, animals,
1989; Viswanath et al. 1997; Lilley et al. 1998). and plants (Glass and Donaldson 1995; Einsele et
In some advanced lesions, fungal hyphae invade al. 1997; Bretagne et al. 1998; Weiland 2000; Bak-
the abdominal viscera, kidney, liver, or spinal cord an et al. 2002). Fungal fish diseases have also been
in several fish species and this almost certainly the investigated, and PCR techniques are used to iden-
cause of death (Chinabut 1990; Wada et al. 1994; tify fish pathogenic A. invadans (A. piscicida; Lil-
Lilley et al. 1998; Viswanath et al. 1998) ley et al. 2003; Phadee et al. 2004) but have not
In general, the taxonomy of the genus Aphan- reported the effectiveness of PCR for detecting A.
piscicida in diseased fish. Only a limited number
of species-specific primers have been described.
* Corresponding author: hatai@scan-net.ne.jp Their specificity and sensitivity must be evaluated
Received September 16, 2003;; accepted August 4, 2004 before they can be widely accepted for routine use.
220
APHANOMYCES PISCICIDA DETECTION USING PCR 221
In the present study, the primer pairs from ITS the dorsal fin with a 25-gauge sterilized needle and
regions were designed for the detection and iden- 1 mL syringe. Control fish were inoculated with
tification of A. piscicida. The ribosomal DNA 100 mL of sterilized water at the same site as in
(rDNA) ITS genes are the widely sequenced DNA test fish. The inoculated fish were reared at room
regions used for taxonomic analyses of fungi. It has temperature (258C) and were collected for DNA
typically been used for molecular systematics at the extraction after the fish showed clinical signs of
species level, and even within species (Carbone and infection. The fungal-infected fish samples were
Kohn 1993; LoBuglio et al. 1993; Hopple and Vil- also confirmed by wet mount observation, reiso-
galys 1994; Rehner and Uecker 1994; Berbee et al. lation, and histopathological examination to en-
1995; Liu et al. 1995, 1997; Yen et al. 1995; Shi- sure that the disease was caused by A. piscicida.
nohara et al. 1999), because of its higher degree of DNA preparation.—For DNA extraction of cul-
variation and because ITS-based sequences have tured hyphae, fungi were incubated at 258C in GY
less selective pressure than other genetic regions of broth for 4 d (Aphanomyces spp.), 3 d (Saprolegnia
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rDNA (Molina et al. 1995; Shinohara et al. 1999). and Achlya spp.), or until young mycelia reached
To supplement available information and to design 0.5–1.0 cm in diameter. Mycelia were washed
species-specific primers for the identification of A. twice with phosphate buffered saline (PBS). They
piscicida, we sequenced the ITS1, 5.8S, and ITS2 were then placed onto tissue paper for drying, and
genes of A. piscicida NJM 0204 (AY283640). The 20–50 mg of mycelia was each transferred to 1.5
sequence showed that the ITS regions of fish-path- mL microcentrifuge tubes. The samples were fro-
ogenic Aphanomyces (AY283640) genes differed zen at 2858C for DNA extraction. DNA extraction
from those of non-fish-pathogenic Aphanomyces from all samples was performed using a genomic
spp. 84-1240 (AF396683). Therefore, the species- prep cell and tissue DNA isolation kit (Amersham
specific primers were designed based on noncon- Pharmacia Biotech, Inc.) according to manufac-
served regions for PCR to develop fast, practical turer’s instructions. The DNA was dissolved in
methods for the detection and identification of sterilized, double-distilled water and DNA con-
Aphanomyces spp. 84-1240 that cause MG in gold- centrations were calculated by the GeneQuant
fish Carassius auratus. proRNA/DNA calculator (Amersham Pharmacia
Biotech, Cambridge, England). The DNA solution
Methods was stored at 48C until used.
Fungal strains and culture.—Forty-two isolates Experimentally infected fish were dissected, and
of Aphanomyces spp., 10 isolates of Achlya spp., infected tissues invaded by A. piscicida were re-
1 isolate of Dictyuchus sp., 4 isolates of Lageni- moved for DNA isolation. Tissue from the lesions
dium spp., and 18 isolates of Saprolegnia spp. were was dissected, and 25–50 mg of each slice was
used in this study (Tables 1, 2). The isolates of transferred into 1.5 mL microcentrifuge tubes and
Aphanomyces spp. were divided into 20 fish-path- frozen at 2858C for DNA extraction. In addition,
ogenic strains and 22 non-fish-pathogenic strains. control tissues (noninjected fish and control fish
All fungi were inoculated onto glucose–yeast ex- injected with sterilized water) were subjected to
tract agar (GY agar) containing 1% glucose, 0.25% DNA extraction. The DNA isolation methods were
yeast extract (Difco Laboratory), and 1% agar, and carried out following the manufacturer’s instruc-
incubated at 258C. tions as described above.
Laboratory induction of mycotic granulomato- DNA sequencing and primer design.—The se-
sis.—Twelve strains of A. piscicida (NJM 9510, quences of ITS1, 5.8S, and ITS2 genes of the A.
RF 6, NJM 9701, NJM 9801, NJM 9901, NJM piscicida NJM 0204 (AY283640) fragment were
0003, NJM 0006, NJM 0008, NJM 0014, NJM amplified using the primers designed from the
0015, NJM 0202, and NJM 0204) were injected rDNA sequence of Aphanomyces sp. (84–1240)
into goldfish 9–12 g in body weight and 65–70 isolated from menhaden (AF396683). Primer ITS-
mm in body length. Goldfish were divided into an F and ITS-R (59-GTCGTAACAAGGTTTCCGTA-
experimental and a control group, with five fish in 39 and 59-TAGCTTAAGTTCAGCGGGTA-39)
each group. were designed from the end of the 18S and the
All Aphanomyces strains were induced for zoo- beginning of the 28S gene, respectively. Poly-
spore formation according to Wada et al. (1996). merase chain reaction mixtures were performed in
One hundred microliters of zoospore suspension, a 50 mL reaction volume using TaKaRa Ex Taq.
approximately 1 3 104 to 1 3 105 spores/mL, were Polymerase chain reaction products were purified
inoculated into the left dorsal trunk muscle under using GFX PCR DNA and gel purification kit
222 PHADEE ET AL.
(Amersham Pharmacia Biotech, Inc., New Jersey, scribed by Sambrook and Russell (2001). The
USA) and cloned into the plasmid vector pCR2.1 primer ITS11 (59-GCCGAAGTTTCGCAAGAA
using the TA cloning kit according to the manu- AC-39) and ITS23 (59-CGTATAGACACAAGCA
facturer’s protocol (Invitrogen, Leek, Nether- CACCA-39) were designed as forward and reverse
lands). Five clones of A. piscicida were sequenced primers, respectively.
using automatic sequencing (400 L; LI-COR) with Polymerase chain reaction amplification.—Poly-
protocols supplied by the manufacturer. The com- merase chain reaction amplification of pure cul-
puter program Genetyx was employed to analyze tured fungal DNA was carried out in a TaKaRa
the sequences. The primers for use in this study PCR thermal cycler personal (TaKaRa Biochem-
were designed from the ITS1 and ITS2 regions of icals) using primers ITS11 and ITS23. Polymerase
A. piscicida NJM 0204 following the method de- chain reaction mixtures were performed in a 25
APHANOMYCES PISCICIDA DETECTION USING PCR 223
TABLE 2.—Achlya spp. (10 isolates), Dictyuchus spp. (1 isolate), Lagenidium spp. (4 isolates), and Saprolegnia spp.
(18 isolates) used in this study; ND 5 no data.
Lagenidium
L. thermophilum NJM 9833 Mud crab Scylla serrata 1998 Indonesia
L. thermophilum NJM 0031 Black tiger shrimp larva Penaeus monodon 2000 Thailand
L. thermophilum NJM 0144 Black tiger shrimp larva 2001 Thailand
L. myophylum NJM 8601 Kuruma shrimp P. japonicus 1986 Japan
Saprolegnia
S. diclina NJM 0009 Ayu 1985 Japan
S. parasitica ATCC 90213 Brook trout Salvelinus fontinalis 1986 Japan
S. diclina ATCC 90215 Coho salmon Oncorhynchus kisutch 1986 Japan
S. diclina NJM 9101 Ayu 1991 Japan
S. salmonis NJM 9851 Sockeye salmon O. nerka 1998 Japan
S. australis NJM 9852 Chum salmon egg O. keta 1998 Japan
S. salmonis NJM 9858 Masu salmon O. masou 1998 Japan
S. parasitica NJM 9868 Sockeye salmon 1998 Japan
S. parasitica NJM 9877 Chum salmon 1998 Japan
S. parasitica NJM 9880 Sockeye salmon 1998 Japan
S. diclina NJM 9908 Masu salmon 1999 Japan
S. sp. NJM 0001 Guppy 2000 Thailand
S. sp. NJM 0128 Ayu 2001 Japan
S. parasitica NJM 0129 Ayu 2001 Japan
S. sp. NJM 0201 Striped snakehead 2002 Thailand
S. semihypogyna IA 1424 Water 1998 Japan
S. ferax ATCC 36146 Water ND England
S. hypogyna ATCC 52721 Water ND England
mL reaction volume containing 0.6 U Taq DNA genes of A. piscicida, identified by sequence
polymerase (Promega), 1.5 mM MgCl2, 0.2 mM matching 100%, was included to ensure that the
deoxynucleotide triphosphate, 0.5 mM of each PCR reaction occurred properly (positive control).
primer, and 20 ng of DNA template (final amount The PCR products were electrophoresed on a 2%
5 1 ng) in thermophilic DNA polymerase 10 3 agarose gel containing 0.5 mg/mL of ethidium bro-
buffer supplied with commercially available Pro- mide. Five microliters of PCR product and 1 mL
mega Taq. The resulting mixture was adjusted to of loading dye were loaded into each lane. A DNA
a final volume of 25 mL by the addition of ster- marker was also run in parallel to approximate the
ilized, double-distilled water. Conditions for PCR size of the PCR products and gels were photo-
involved preheating for 5 min at 948C, followed graphed on an ultraviolet illuminator.
by 25 cycles of amplification of the target se- DNA amplification of fungal-infected fish tis-
quence. Each cycle consisted of a denaturation at sues involved two replicates of each strain of A.
948C for 30 s, annealing at 658C for 30 s, and piscicida. All amplification procedures were as
primer extension at 728C for 1 min, with 5 min pure cultured fungus, except that for the DNA tem-
for a final extension at 728C. Additionally, control plate, the final amount used was 5 ng and 35 cycles
amplifications using primers only were performed of PCR amplification were carried out. Also, a
to ensure that reagents used were not contaminated positive control was performed with pure cultured
with extraneous template DNA (negative control). DNA of A. piscicida NJM 0204, and negative con-
One nanogram of plasmid clone containing the ITS trols consisted of reaction components without
224 PHADEE ET AL.
FIGURE 1.—Agarose gel showing the PCR products of amplified genomic DNA from Aphanomyces spp. using
a specific primer (ITS11 and ITS23). The numbers in the left margin indicate the positions of size markers in base
pairs (bp). The leftmost lane shows a 100-bp ladder; lane C is the negative control, with no DNA template; lanes
1–23 correspond to the detection of the following strains of Aphanomyces spp.: (1) 3 P, (2) 4 P, (3) NJM 9201, (4)
NJM 9510, (5) RF 6, (6) NJM 9701, (7) NJM 9801, (8) NJM 9803, (9) NJM 9901, (10) NJM 9903, (11) NJM
9907, (12) NJM 0002, (13) NJM 0003, (14) NJM 0004, (15) NJM 0006, (16) NJM 0008, (17) NJM 0014, (18)
NJM 0015, (19) NJM 0202, (20) NJM 0204, (21) 84–1240, (22) RAR 35–9, and (23) D3.
Downloaded by [Florida Atlantic University] at 11:46 10 November 2014
template DNA from noninjected goldfish tissue Specificity of Polymerase Chain Reaction Primers
and DNA from goldfish injected with sterilized The DNA from 76 strains of Saprolegniaceae
water. were used as DNA templates for PCR amplifica-
Minimal concentration of fungal DNA.—The tion of the primer pairs (ITS11 and ITS23), in-
minimal amount of fungal DNA for successful de- cluding 20 isolates of fish-pathogenic and 22 iso-
tection by PCR amplification with the primer set lates of non-fish-pathogenic Aphanomyces, 10 iso-
(ITS11 and ITS23) was determined. This was done lates of Achlya, 1 isolate of Dictyuchus, 4 isolates
using the DNA of A. piscicida from cultural hyphae of Lagenidium, and 18 isolates of Saprolegnia spe-
and the muscle tissues of goldfish experimentally cies. The PCR procedure using ITS11 and ITS23
infected with A. piscicida NJM 0204. Polymerase detected only 20 isolates of fish-pathogenic
chain reaction amplification was performed as de- Aphanomyces spp. that exhibited homologous am-
scribed above, with a selected DNA concentration plicons of 550 bp as same as the positive control.
between 1 fg and 1 ng of cellular DNA and 2.5 fg Two strains of Aphanomyces isolated from men-
to 5 ng of infected tissues. haden Brevoortia spp. (84-1240 and RAR 35-9)
were not detected (Table 3; Figure 1). However,
Results there were no PCR products from the negative con-
trol, the non-fish-pathogenic Aphanomyces spp.,
Sequence Analysis of Aphanomyces piscicida
Achlya spp., Dictyuchus sp., Lagenidium spp., and
Sequencing of A. piscicida NJM 0204 resulted Saprolegnia spp. (Table 3).
in a 735-base-pair (bp) fragment including a partial
sequence of an 18S rDNA fragment (bp 1–43); Detection of Aphanomyces piscicida in Goldfish
complete sequences of ITS1 (bp 44–201), 5.8S Muscle
rDNA (bp 202–361), and ITS2 (bp 362–692); and The experimental goldfish began to develop
a partial sequence of 28S rDNA (bp 693–735). The clinical signs of MG 5–10 d after inoculation with
GenBank accession number of the sequence is A. piscicida, although the time to lesion develop-
AY283640. The present sequence was aligned with ment varied among strains. The diseased fish grad-
non-fish-pathogenic Aphanomyces spp. 84-1240, ually became ulcerated and scale displacement was
accession number AF396683; the alignment score observed. The lesions around the injected area
showed 78.3% homology. It was found that the were generally necrotic and there was hemorrhage;
sequence in this study corresponded to the ITS1 fungal hyphae were seen erupting from lesions at
and ITS2 regions of a non-fish-pathogenic strain, the site of injection among scales, and dermal ul-
although their 5.8S sequence slightly differed, cers were seen in some fish (Figure 2a). External
while the 18S and 28S rDNA regions were highly fungus was not observed, and no fish injected with
conserved. Therefore, the primer pair used in this sterilized water developed lesions. After the ex-
study was designed from the nonconserved area perimentally infected fish developed severe lesions
of the ITS1 and ITS2 regions, designed as ITS11 (10–20 d postinjection), tissues were collected.
and ITS23, respectively. Fungal hyphae from fish muscle and morpholog-
APHANOMYCES PISCICIDA DETECTION USING PCR 225
FIGURE 3.—Agarose gel showing the PCR products of amplified genomic DNA of A. piscicida2infected goldfish
tissue using a specific primer (ITS11 and ITS23). The numbers in left margin indicate the positions of size markers
in base pairs. The leftmost lane shows a 100-bp ladder; lane C1 is the negative control, with no DNA template;
lane C2 is the positive control, with genomic DNA of A. piscicida NJM 0204; lane 1 is the genomic DNA of
Downloaded by [Florida Atlantic University] at 11:46 10 November 2014
noninjected goldfish; lane 2 is the genomic DNA of goldfish injected with sterilized water; lanes 3–14 correspond
to the following strains of A. piscicida with goldfish tissue DNA: (3) NJM 9510, (4) RF 6, (5) NJM 9701, (6) NJM
9801, (7) NJM 9901, (8) NJM 0003, (9) NJM 0006 (10) NJM 0008, (11) NJM 0014, (12) NJM 0015, (13) NJM
0202, and (14) NJM 0204.
spp., where amplification of the characteristic band cicida (seven isolates) from various countries and
was not seen. We found, however, that these prim- non-fish-pathogenic Aphanomyces strains (13 iso-
ers were unable to detect some fish isolated strains lates) were examined (accession numbers
(84-1240 and RAR 35–9). These strains are sap- AY283641-8 and AY455771-7). It was found that
rophytic Aphanomyces spp. based on the morpho- the ITS1, 5.8S, and ITS2 regions of all A. piscicida
logical, biological, genetic and serological char- strains were identical, and that the non-fish-path-
acteristics of these strains, which were different ogenic Aphanomyces isolates differed from the
from other strains of Aphanomyces isolated from fish-pathogenic Aphanomyces and also from each
infected fish (Dykstra et al. 1989; Willoughby et other (data not shown). These results suggest that
al. 1995; Lilley and Roberts 1997; Lilley et al. A. piscicida can be differentiated from other oom-
1997; Kurata et al. 2002, Lilley et al. 2003). How- ycetes by sequencing the ITS1, 5.8S, and ITS2
ever, these strains did not show pathogenicity to regions. Lilley et al. (2003) have differentiated A.
goldfish, a fish species susceptible to A. piscicida invadans from other fungi and have shown it as a
(Phadee et al., Nippon Veterinary and Animal Sci- single fish-pathogenic species by restriction frag-
ence University, unpublished data). Furthermore, ment length polymorphism analysis of rDNA and
to confirm the results, DNA sequences of A. pis- by sequencing the ITS1 regions.
Polymerase chain reaction was effective in de-
TABLE 4.—Detection of goldfish with experimentally tecting A. piscicida from goldfish tissue DNA and
induced mycotic granulomatosis using the ITS11 and showed homologous amplicons with pure cultured
ITS23 primer set. A. piscicida. In addition, we have been successful
Species Isolate Resulta in using PCR to detect the pathogen in naturally
occurring MG in Ayu (unpublished data). Fur-
Intact fish 2
Control b 2 thermore, the primer set (ITS11 and ITS23) de-
Aphanomyces piscicida NJM 9510 1 tected 250 fg and 500 fg as the minimum concen-
A. invadans RF 6 1 tration of pure culture fungi and fungus with fish
A. piscicida NJM 9701 1
A. piscicida NJM 9801 1 DNA, respectively. In previous studies, 1 pg of
A. piscicida NJM 9901 1 primer set P1 and P2 was sufficient to detect A.
A. piscicida NJM 0003 1 astaci cellular DNA (Oidtmann et al. 2002). Our
A. piscicida NJM 0006 1
A. piscicida NJM 0008 1 results indicate that the primer set in this study is
A. piscicida NJM 0014 1 useful in detecting MG and can differentiate fish-
A. piscicida NJM 0015 1 pathogenic Aphanomyces spp. from other similar
A. piscicida NJM 0202 1
A. piscicida NJM 0204 1 fungi. However, at the onset of disease or low lev-
a
els of infection, this procedure may not be suitable
Plus signs indicate detection of the condition, minus signs no
detection. because PCR analysis depends on the amount of
b Goldfish injected with sterilized distilled water. fungal DNA template. Nested PCR or some other
228 PHADEE ET AL.
rapid diagnosis method (such as fluorescent anti- sequence divergence within internal transcribed
body technique) may be necessary for the detec- spacer 1 of the Sclerotiniaceae. Mycologia 85:415–
427.
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