The Veterinary Journal 197 (2013) 489–491

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The Veterinary Journal

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Short Communication

Detection of Neorickettsia risticii from various freshwater snail speciescollected from a district irrigation canal in Nevada County, California

1090-0233/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.tvjl.2013.02.024

⇑ Corresponding author. Tel.: +1 530 754 5523.E-mail address: npusterla@ucdavis.edu (N. Pusterla).

Nicola Pusterla a,⇑, Daniel Hagerty a, Samantha Mapes a, Josh VanGeem a, Lindsey T. Groves b,Mario Dinucci c, Langdon C. Fielding d, Jill C. Higgins d

a Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 95616, USAb Natural History Museum of Los Angeles County, Los Angeles, CA 90007, USAc Sierra Equine, Grass Valley, CA 95945, USAd Loomis Basin Equine Medical Center, Loomis, CA 95650, USA

a r t i c l e i n f o

Article history:Accepted 26 February 2013

Keywords:Neorickettsia risticiiEquine neorickettsiosisHorseFreshwater snailsIrrigation canalPotomac horse fever

a b s t r a c t

This study investigated the role of a district irrigation canal in Nevada County, California, USA, as thepoint source of infection for Neorickettsia risticii, causative agent of equine neorickettsiosis (EN). A totalof 568 freshwater snails comprising Juga spp., Planorbella subcrenata (Carpenter, 1857) (Rough Rams-horn), Physella virgata (Gould, 1855) (Protean Physa) and feces from three horses with EN were collectedand tested for N. risticii by real-time PCR. A total of four freshwater snails tested PCR positive for N. risticii.Phylogenetic analysis showed 99.8–100% homology between the different snail and horse N. risticii iso-lates. This study represents the first report of infection with N. risticii in Planorbella subcrenata and sug-gests that the irrigation canal was the aquatic environment responsible for the spread of N. risticii.

� 2013 Elsevier Ltd. All rights reserved.

Neorickettsia risticii is the etiologic agent of equine neoricketts-iosis (EN), or Potomac horse fever (PHF), an acute enterocolitis ofhorses. Neorickettsia risticii has recently been associated with trem-atode-vectors, which use freshwater snails and aquatic insects asintermediate hosts and insectivorous birds and bats as definitivehosts (Pusterla et al., 2003; Gibson et al., 2005). In Northern Cali-fornia, the species of snail incriminated in the life cycle of the hel-minth vector infected with N. risticii has been identified as Jugayrekaensis (Henderson, 1935, [Yreka juga]), a common pleuroceridsnail, which inhabits fresh or brackish stream water in the North-western United States (Barlough et al., 1998). Additionally, DNAfrom N. risticii has been detected in virgulate cercariae in lymnaeidsnails (Stagnicola spp.) from Northern California, in virgulate xiph-idiocercariae isolated from pleurocerid snails (Elimia livescens,(Menke, 1830), [Liver Elimia]) in central Ohio, and from pleuroce-rid snails (Elimia virginica, (Say, 1817), [Piedmont Elimia]) in cen-tral Pennsylvania, indicating that other species of freshwatersnails may also harbor infected trematodes (Reubel et al., 1998;Kanter et al., 2000).

In recent years, there have been an increasing number of re-ported EN cases from Nevada County, California. Of epidemiologi-cal interest is the fact that water is supplied to the premises whereconfirmed EN cases have historically been diagnosed through a dis-

trict irrigation canal system. The aim of this study was, therefore,to determine if freshwater snails collected from these canals har-bored N. risticii.

A total of 568 freshwater snails, including 218 Juga spp., 140Planorbella subcrenata and 210 Physella virgata (Fig. 1), werecollected from five different sites along the irrigation canal fromSeptember 2010 to August 2011 (Fig. 2). Each snail was extractedfrom its shell using clean forceps and scalpel blades, and placedinto a 96-well plate containing 500 lL of lysis solution. All extrac-tion steps were performed in a hood with laminar flow. ProteinaseK and two grinding beads (4 mm diameter, stainless steel beads,SpexCertiprep) were added and the tissues homogenized in aGenoGrinder2000 (SpexCertiprep) for 2 min at 1000 strokes perminute.

Protein digest was at 56 �C for 30 min followed by a 30 min per-iod at �20 �C to reduce foam. Two hundred microliters of the mix-ture were processed for DNA purification using an automatedextraction protocol as previously reported (Pusterla et al., 2000a).Extracted nucleic acids were tested for the presence of N. risticiiby real-time PCR using a previously established and validated pro-tocol (Pusterla et al., 2000a). Known positive and negative controlswere used with every PCR run. Furthermore, a real-time PCR assaytargeting the universal eukaryotic 18S rRNA gene was used asquality control (i.e. efficiency of DNA purification and amplifica-tion) and as an indicator of inhibition (Eukaryotic 18S rRNA Endog-enous Control, Applied Biosystems).

Fig. 1. (a) Juga species, hypotype LACM 176641, 15.1 mm (shell maximum height).(b) Physa virgata (Gould, 1855), hypotype LACM 176643, 4.9 mm (shell maximumheight). (c) Apertural view; (d) apical view and (e) umbilical view of Planorbellasubcrenata (Carpenter, 1857), hypotype LACM 176642, 8.1 mm (shell maximumwidth).

Fig. 2. Map of Nevada County in California, USA, showing the irrigation district,

490 N. Pusterla et al. / The Veterinary Journal 197 (2013) 489–491

Three horses diagnosed with PHF from different localities in Ne-vada County were used for phylogenetic analysis. A 527 nucleotidesegment of the 16S rRNA gene of N. risticii was generated from thesnail and horse isolates in order to determine phylogenetic rela-tionship (Reubel et al., 1998).

Nucleic acid extraction was successful in all 568 freshwatersnails based on the detection of the 18S rRNA gene. Four freshwa-ter snails tested PCR positive for N. risticii. These included one Jugaspecies (0.4%) and three P. subcrenata (2.1%) snails, but none of theP. virgata snails tested PCR positive for N. risticii. All three horsespresented to Loomis Basin Equine Medical Center with classicalsigns of EN, including depression, anorexia, fever, colic and soft-formed feces tested PCR positive for N. risticii in feces (Bertinet al., 2011). Sequence comparison of a 527 nucleotide segmentof the 16S rRNA gene of N. risticii showed 100% homology amongstthe snails and between 99.3% and 100.0% homology with the threehorses from Nevada County (Fig. 3).

The biology of N. risticii is associated with an aquatic environ-ment, which explains why EN cases have a seasonal occurrenceand are commonly observed near waterways in endemic areas(Barlough et al., 1998). This study reports on the detection of N. ris-ticii in freshwater snails and horses from Nevada County, Califor-nia. This newly recognized endemic area is geographicallydistinct from other endemic areas in California, which are locatedclose to the Oregon border. In the rural area of Nevada Countywater is provided via a canal system from April to October andused mainly for local agriculture, including pasture irrigation.The water originates from Scotts Flat Reservoir and is carried inan open canal system for over 150 miles (ca. 241 km) to approxi-mately 32,000 irrigable acres (ca. 13,000 ha). To the knowledgeof the authors, this is the first report associating a district irrigationcanal system with the spread of N. risticii.

sites of snail collection and horses diagnosed with equine neorickettsiosis.

Fig. 3. Phylogenetic relationship of field strains of N. risticii from snails and horses with PHF from California and other endemic areas of the United States (Siskiyou County,California; Ohio; Illinois, Oregon). The field isolates are labeled by species (snail or horse) and location (County or State). Analyses were done using the MEGA 5.10 versionBeta 4 software package.

N. Pusterla et al. / The Veterinary Journal 197 (2013) 489–491 491

Demonstrating endemic occurrence of N. risticii was based onthe detection of the neorickettsial organism in freshwater snailsand resident horses with signs compatible with EN. Previous workhas shown that the number of PCR positive snails appears to de-pend on the size of the snails, the month of collection, and geo-graphic origin (Pusterla et al., 2000b). In the present study, wedetected a prevalence of infected freshwater snails ranging from0.4% to 2.1% which is in agreement with previous reports (Barloughet al., 1998; Pusterla et al., 2000b). Juga yrekaensis is considered themain species of freshwater snails associated with the life cycle ofthe digenetic trematode vectors of N. risticii in Northern California(Reubel et al., 1998). This was in contrast with the present study,where the majority of the PCR positive freshwater snails belongedto P. subcrenata. This planorbid snail is common in the Northwest-ern United States and can be recognized by its sinistrally-coiledshell (Burch, 1989). Previous work has shown that in endemicareas various freshwater snail species can harbor N. risticii (Reubelet al., 1998).

EN was diagnosed in the horses from Nevada County based onclinical signs, laboratory detection of N. risticii via PCR and re-sponse to treatment with oxytetracycline and supportive care aspreviously reported (Bertin et al., 2011). All the affected horseshad direct access to irrigated pastures and lived 100–300 m fromthe nearest irrigation canal. The close relationship between fresh-water snail and horse N. risticii isolates was further supported bythe high sequence homology of the partial sequence of the 16SrRNA gene of N. risticii (Reubel et al., 1998). The study representsthe first report of infection with N. risticii in Planorbella subcrenataand suggests that the irrigation canal was the aquatic environmentresponsible for the spread of N. risticii.

Conflict of interest statement

The authors have no financial or personal relationship withother people or organizations that could inappropriately influenceor bias the content of the paper.

Acknowledgement

The authors wish to thank Edward J. Johannes (Deixis Consul-tants, SeaTac, WA) for confirming the identification of the Jugaspecimens.

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