Ann. N.Y. Acad. Sci.

990: 239–247 (2003). ©2003 New York Academy of Sciences.

Identification of Trematode CercariaeCarrying

Neorickettsia risticii

in Freshwater Stream Snails

BAE-KEUN PARK, MEEN-JU KIM, EUN-HA KIM,

a

MIN-SEOK KIM,

a

DONG-GYUN NA,

a

AND JOON-SEOK CHAE

a

College of Veterinary Medicine, Chungnam National University, Yousung-gu, Taejeon 305-764, Korea

a

College of Veterinary Medicine, Chonbuk National University,Jeonju, Jeonbuk 561-756, Korea

A

BSTRACT

: We provide evidence of

Neorickettsia

(

Ehrlichia

)

risticii

Holland,the agent of Potomac horse fever, in trematode larval stages found in aquaticsnails and insects collected from a stream in Korea, using nested polymerasechain reaction (PCR) and sequence analysis of the 16S rRNA gene fragmentamplified from several cercaria species. It was observed that of 423 (13.1%) of3,219 snails infected with cercariae, 77 (20.8% of the 371) were infected with

N. risticii

. Five families of trematode cercariae, Schistosomatidae, Echimosto-matidae, Heterophyidae, Microphallidae, and Acanthocopidae were identifiedmorphologically within

Semisulcospira libertina

,

Radix auricularia coreana,

and

S. gottschei

snails.

Echinostoma cinetorchis

,

E. hortense

, and

Metagonimus

sp. were identified based on both the cercarial morphology as well as by phy-logenetic analysis of the amplified 18S rRNA gene sequences. Adult aquaticinsects were also collected from the same sites and were sorted into five species,

Ischnura asiatica

in Coenagrionidae and

Calopteryx japonica

,

Sympetrum dar-winianum

,

Symptrum eroticum

, and

Symptrum parvulum

in Calopterygoidae.One thousand and two hundred eighty five metacercariae (classified intogroups A through F) were isolated from 310 adult aquatic insects, and theaverage number of metacercariae per aquatic insect was 4.1. However, therewas no amplification of

N. risticii

from these metacercariae.

K

EYWORDS

:

Ehrlichia risticii

; aquatic snail; aquatic insect; metacercariae;cercaria

INTRODUCTION

Potomac horse fever is caused by the rickettsial organism

Ehrlichia risticii

Hol-land. The disease was first recognized in 1979 in pastures along the Potomac Riverand has been reported in many regions of the United States, Canada, and possiblyEurope.

1

−

5

Despite of the intensive research efforts, the transmission of Potomachorse fever has remained a mystery because the life cycle of the transmissional agentand the mode of transmission are unknown. Part of the mystery has been recently

Address for correspondence: Joon-Seok Chae, College of Veterinary Medicine, ChonbukNational University, Jeonju, Jeonbuk 561-756, Korea. Fax: +82-63-270-3778.

jschae@moak.chonbuk.ac.kr

240 ANNALS NEW YORK ACADEMY OF SCIENCES

uncovered through the detection of

Neorickettsia risticii

in trematodes that infestoperculate freshwater snails. The presence of

N. risticii

DNA was initially detectedby PCR in freshwater snails (Pleuroceridae:

Juga

spp.) in northern California.

6

Sub-sequently, the presence of

N. risticii

has been demonstrated in virgulate cercariaeinfecting the snails by amplification and sequence analyses of 16S rRNA, 51 kDamajor antigen, and groEL heat shock protein gene fragment.

7

Chae and colleagues

8

have isolated

N. risticii

from the trematode stage in aquatic snails and accomplishedexperimental transmission of

N. risticii

to horses and mice.

8,9

These results suggestthat trematodes may be involved in the transmission of Potomac horse fever.

Trematodes belong to Digenea, which has five larval stages in the life cycle: mira-cidium, sporocyst, redia, cercaria, and metacercaria. Some of these stages may occurin a wide range of other invertebrates and sometimes in vertebrates, which serve asan intermediate host. Snails are the first intermediate host of digenetic trematodes.Cercaria is produced by the sporocyst or the redia in snails. Virgulate cercariae areassociated with lecithodendriid trematodes; and in the genera described, snails andaquatic insects act as intermediate hosts.

10,11

Stonefly nymphs and caddisfly larvaeare the most important second intermediate host for several species of lecithodendriidtrematodes, such as

Allassogonoporus vespertilionis

Macy and

Acanthatrium ore-gonense

Macy, the common trematodes of bats.

10

In this study, we provide molecularevidence of the presence of

N. risticii

, the agent of Potomac horse fever, in trematodelarval stages found in aquatic snails and insects collected from a stream in Korea,using nested polymerase chain reaction (PCR) and sequence analyses of the 16SrRNA gene fragment amplified from several cercaria and metacercaria species.

MATERIALS AND METHODS

Collection of Snails

Freshwater snails and aquatic insects were collected in September and October2001 by hand and net from streams located in the Chungcheong and Jeonra provinc-es, Korea. Two species of the collected snails,

Semisulcospira libertina

and

Radixauricularia coreana

, were broken up in 0.45% saline with sterile scissors and twee-zers. The intestine and midgut gland of snails were carefully chopped to allowdestruction of the snail tissue and release of the cercariae. Under a stereomicroscopethe cercariae were liberated from the snail material and collected using Pasteurpipettes into tubes and stored in

−

70

°

C for PCR and sequencing. The collected cer-cariae were identified based on the cercarial morphology as well as phylogeneticanalysis of the amplified trematode 18S rRNA gene fragment sequences.

Collection of Adult Flies

For the isolation of metacercaria from adults of dragonfly (

Sympetrum darwin-ianum

,

S. eroticum

,

S. depressiusculum

), the wings of dragonfly were removed,and the body was split in pieces and then digested with artificial gastric juice (6 g ofpepsin and 7 mL of HCl in 1 L distilled water) in a beaker at 37

°

C for 3 hours. Thedigest was collected with distilled water and transferred into a measuring cylinderand undigested material allowed to sediment for 1 hour. The supernatant was then

241PARK

et al.

:

NEORICKETTSIA RISTICII

IN FRESHWATER SNAILS

discarded by aspiration. This process was repeated three times to clarity. The super-natant was placed in a Petri dish and the metacercariae were harvested in 0.45%saline under a stereomicroscope using a Pasteur pipette, identified under the lightmicroscope, and stored at 4

°

C refrigerator until further study. Twenty metacercariaewere fed to each of the quails using a gastric needle for identifying adult trematode.Fecal examination for trematode egg production was performed every 24 h for thenext 5 days.

DNA Extraction

DNA extraction was done using a QIAamp tissue kit (Qiagen, Inc., Chatsworth,CA) according to the provided protocol. The DNA concentration was determined onthe basis of reading of A

260

and A

280

with GeneQuant

pro

spectrophotometer (Amer-sham Pharmacia Biotech, Inc).

PCR Amplification of Trematode 18S rRNA Gene Fragment

Amplification of trematode 18S rRNA gene fragments from all other specieswas carried out by PCR using primers SHF 5

′

-acgggtgcatttattagaac-3

′

and SHR5

′

-agcactcaaatttgttcaaagt-3

′

designed from the sequence of

S. haematobium

. PCRreactions were performed in 20 volumes of 10

×

buffer containing 2.5 mM MgCl

2

,0.2 mM dNTP, 10 pmol of each primer, 200 ng of template DNA, and

Taq

poly-merase (2.5 U; Promega). PCR products were purified by GGX

TM

PCR DNA Puri-fication Kit (Amersham Pharmacia Biotech Inc).

PCR Amplification of

N. risticii

16S rRNA Gene Fragment

N. risticii

was detected in cercariae by amplifying the 16S rRNA gene fragmentby nested PCR as previously described.

12

DNA Sequencing

Amplified PCR products of the trematode 18S rRNA gene fragments weresequenced by a dideoxy chain termination reaction that was detected with a ABIPRISM 3700 DNA Analyzer. Nucleotide sequence homology searches were madethrough the National Center for Biotechnology Information BLAST network service.Comparisons of sequence homology were made with the ALIGN query (GeneStream,http://www2.igh.cnrs.fr/bin/align-guess.cgi). Multiple-sequence alignments weremade by Vector NTI Suit program (v. 5.2.1.3, InforMax, Inc, USA).

RESULTS

Five families of trematode cercariae (Echinostomatidae, Schistosomatidae,Heterophyidae, Microphallidae, and Acanthocopidae) were identified from

Semi-sulcospira libertina

and

Radix auricularia coreana

snails (see F

IGURE

1). Twospecies of Echinostomatidae (

Echinostoma cinetorchis

and

E. hortense

) were iden-tified from

Radix auricularia coreana

both morphologically and by phylogeneticanalysis of the sequence amplified using 18S rRNA.

E. cinetorchis

,

E. hortense

, and

242 ANNALS NEW YORK ACADEMY OF SCIENCES

Metagonimus yokogawai

were identified based on both the cercarial morphology aswell as by phylogenetic analysis of the amplified trematode 18S rRNA genesequences (see F

IGURE

2). The trematode cercariae infection ratio in the 423 cercar-iae observed from 3,219 snails was 13.1% (see T

ABLE

1).Adult aquatic insects were also collected from the same sites and were sorted into

five species,

Ischnura asiatica

in Coenagrionidae and

Calopteryx japonica

,

Sym-petrum darwinianum

,

Symptrum eroticum

, and

Symptrum parvulum

in Caloptery-goidae. No meatacercaria were isolated from the 633 adults of

Ischnura asiatica

FIGURE 1. Phylogenetic tree of cercariae from aquatic snails in Korea.

243PARK

et al.

:

NEORICKETTSIA RISTICII

IN FRESHWATER SNAILS

(data not shown). A few metacercariae of Plagiorchidea and Prosthogonimidae weredetected in the dragonfly and the most of metacercaria were species of Lecithoden-driidae. The metacercariae of Plagiorchidea and Prosthogonimidae were not studiedin this study. One thousand and two hundred eighty five metacercariae (classifiedinto groups A through F) were isolated from 310 adults of Calopterygoidae, and anumber of average metacercariae per aquatic insect was 4.1 (see F

IGURE

3 andT

ABLE

2). The harvested metacercariae, grouped A to F by morphology, were mainlyof the groups D and F. The size range was 219.88

−

310.64

×

193.96

−

283.00

µ

m and127.66

−

102.79

×

102.29

−

121.99

µ

m. For identification and

N. risticii

transmissionof adult trematode, the metacercariae (group D and F) were inoculated orally in toeach of five quails. A few eggs were harvested from feces post inoculation day 23(prepatent period) and patent period was 25 days. The egg sizes of group D and Fwere 45.35 x 33.03

µ

m and 96.14 x 68.88

µ

m, respectively (see F

IGURE

4). However,there was no amplification of

N. risticii 16S rRNA gene fragment when the templateDNA was from either the metacercaria from dragonfly or any organs of infectedquails.

DISCUSSION

Our discovery that there is molecular evidence of infection with N. risticii of theseveral cercariae present in snails and aquatic insects indicates that digenial cercar-iae could be important in the epidemiology of Potomac horse fever in several ways:

FIGURE 2. The cercariae from Semisulcospira libertina snails (B and C) and Radixauricularia coreana snails (A and D) in Korea. A, Echinostoma cinetorchis; B, Schistoso-matidae; C, Metagonimus sp.; D, E. hortense.

244 ANNALS NEW YORK ACADEMY OF SCIENCES

TABLE 1. Infection rates of cercariae and N. risticii from snails in Chungnam andJeonra province, Korea

ProvincesSnails

(number of snails)

Cercariae Positive PCR for

N. risticiiSpecies Number of

infected snails (%)

Chungcheong

Semisulcospira libertina (1,502)

Schistosomatidae 69 (4.6) 11

Microphallidae 28 (1.9) 5

E. cinetorchis 36 (1.0) 0

M. yokogawaii 4 (0.3) 0

Trichobilharzia 4 (0.3) 0

E. hortense 10 (0.7) 0

Furcocercus 1 (0.1) 0

Radix auricularia coreana (368)

E. cinetorichis 16 (4.3) 6

E. hortense 36 (9.8) 2

Fasciola sp. 13 (3.5) 1

Schistosomatidae 1 (0.3) 0

S. gottschei (73) E. cinetorchis 5 (6.8) 0

Jeonra S. libertina (1,276)

Furcocercus 19 (1.5) 8

Xiphidiocercaria 139 (10.9) 34

Unknown 52 (4.1) 10

Total 3,219 423 (13.1) 77

FIGURE 3. The metacercariae from dragonfly (Calopterygoidae) in Korea. The har-vested metacercariae were grouped A to F by morphology.

245PARK et al.: NEORICKETTSIA RISTICII IN FRESHWATER SNAILS

by infected cercariae, by serving as infectious prey for reservoir animals that carrythe definitive host of the parasite, or by directly providing a mean of transport of thedisease to the terrestrial habitat when infected insects metamorphose to the wingedstage.

Trematodes belong to Echinostomatidae parasitized in the intestine of birds andmammals and several hundred species are found worldwide. In Korea, seven speciesof Echinostomatidae (Echinostoma revolutum, E. gotoi, E. miyagawai, E. cine-torchis, E. hortense, Echinoparyphium recurvatum, and Echinochasmus japonicus)have been reported.13−15 Three freshwater lymnaeid snail species (R. auriculariacoreana, Austropeplea ollula, and Fossaria truncatula), belonging to the familyLymnaeidae, have been reported in Korea. In this study, only the predominating snailof Korea, R. auricularia coreana, was examined, and we found the cercariae of threespecies E. cinetorchis, E. hortense, and Metagonimus yokogawai. E. cinetorchis, E.hortense, and Fasciola sp. have been previously observed from R. auricularia core-ana. N. risticii was detected by PCR in two species of echinostomatoid trematodes.Frog and tadpole (such as Rana nigromaculata, R. rugosa, R. catesbiana)16 and fish(such as Misgurnus anguillicaudatus, Carassius carassius, Acheilognathus

TABLE 2. Prevalence of metacercariae in adult flies from Chungcheong and Jeonraprovince, Korea

Provinces Species of Adult Flies Number of Adult Flies

Number of Metacercaria

Mean of Infection Numbers

N. risticii PCR

Chung-cheong

Sympetrum darwinianum 98 787 8.0 0

Calopteryx japonica 70 131 1.9 0

Jeonra

Symptrum darwinianum 113 316 2.8 0

Symptrum eroticum 6 0 0 0

Symptrum parvulum 23 51 2.2 0

Total 310 1,285 4.1 0

FIGURE 4. The eggs of trematode from quail infected with metacercariae originatedfrom dragonfly.

246 ANNALS NEW YORK ACADEMY OF SCIENCES

sp., Odontobutis obscura interrupta, and Squalidus coreanus) have been reported assecond intermediate hosts of echinstomatoid trematode and, hence, may transmittrematodes to predators. Thus the ability of trematodes to infect snails, frogs, andfish makes it more likely that some trematodes will find their way into a given defin-itive host.

Semisulcospira libertina (Mesogastropoda: Pleuroceridae) is easily found in thestreams of South Korea and is an important intermediate host of digenial trematodes.But, little data can be found on the ecology of Semisulcospira snails and infestationof the snails with the cercariae of digenial trematodes. Choi17 reported, in snails,seven species of known cercaria (Cercaria incerta, Cercaria nipponensis, Cercariayoshidae, Centrocestus armatus, Metagonimus species, Notocotylus magniovatus,and Paragonimus westermani) and four kinds of undetermined cercariae. The lifecycles of Cercaria incerta, Cercaria nipponensis, Cercaria yoshidae, and Notocot-ylus magniovatus are undetermined. In this study, we think that the cercaria classi-fied Microphallidae may be some of them.

Dragonfly is a second intermediate host of Plagiorchidea, Hematoloechidae,Prosthogonimidae, and Lecithodendriidae.18 In this study, a few metacercariae ofPlagiorchidea and Prosthogonimidae were detected in the dragonfly, but most of themetacercaria were species of Lecithodendriidae and Microphallidae. We presumethat the frog will have an important role in the N. risticii transmission cycle, becausetadpoles and frogs serve as the second intermediate and final host for most speciesof Ehrlinostomatidae and Lecithodendriidae, respectively.

Our findings expand and support the observation that N. risticii is associated withan aquatic environment and that digenetic trematodes are involved in the transmis-sion cycle of N. risticii.

ACKNOWLEDGMENTS

This work was supported by grant No. (R01-2001-00246) from the BasicResearch program of the Korea Science & Engineering Foundation.

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