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IDENTIFICATION OF AN ENZOOTIC DIARRHEA ("Shasta River Crud") IN NORTHERN CALIFORNIA AS POTOMAC HORSE FEVER
Abstract
For at least 25 years, veterinary practitioners in areas of Siskiyou and Shasta counties, California, have recognized a diarrheic disease of horses that is referred to locally as the "Shasta River crud" (SRC). t,2 It is seen in the spring and summer months along the Shasta and Klamath rivers, with the early cases developing nearest the river and later cases occurring progressively farther away. The illness is characterized by signs of anorexia, lethargy, diarrhea, variable fever, and in some cases laminitis, and is responsive to systemic tetracyclines and intensive fluid therapy. Limited serologic testing (immunofluorescence assay, IFA) has provided some evidence that the disease may be linked to Ehrlichia risticii, the agent of Potomac horse fever. 1,a Owing to the remarkably high false-positive rate of the E. risticii IFA, 4 however, as well as an absence of confirmatory findings (e.g., isolation or detection of the causative agent), the etiology of this seasonally-recurring illness has remained unclear.
Here we report the identification ofE. risticii DNA in blood buffy-coat cells from two horses with clinical signs of SRC. The agent was detected by a nested polymerase chain reaction (PCR), 2 and by Southern hybridization using an internal oligonucleotide probeZ; final confirmation of identity was provided by DNA sequencing. Sequencing results further indicated that the SRC agent is most closely related to the Kentucky strain s of E. risticii.
Case No. 1 ("Shorty"). In early May 1996, a 10-9 1 2-+- year-old Quarter Horse gelding living along the Shasta River in Weed, California (Siskiyou County), was examined for an illness of 1-2 days' duration. The horse had resided in the county for only a year and had been vaccinated in the spring for E. risticii by its owner. The horse was alert and eating, with audible gut sounds and a profuse, soft-to-watery diarrhea. The rectaltemperature was 99.5~ the heart rate 44 bpm, and the capillary refill time 2 seconds. Hematologic abnormalities included neutrophilia with a left shift, lymphopenia, and elevated fibrinogen. The horse was treated aggressively with over 30 liters of lactated Ringer's solution i.v., along with oral electrolytes, mineral oil, activated charcoal, non-steroidal antiinflammatory agents, and antibiotics (including systemic oxytetracycline). The gelding had been on antibiotics for about 24 hours at the time a blood sample was drawn for PCR. The diarrhea ceased within 48 hours of initiation of therapy and the horse made an uneventful recovery over the next week.
Case No. 2 ("Shotgun"). In mid-May 1996, a 3-year-old Quarter Horse gelding pastured along the Shasta River in Mt. Shasta, California (Siskiyou County) was examined for an illness of 4 days' duration. Clinical signs included lethargy, anorexia, and diarrhea. Four other horses on the ranch had died with similar signs, several years previously. The present horse had been vaccinated against E. risticii by its owner. Clinical findings included a rectal temperature of 101.8~ a pulse rate of 60, and a respiratory rate of 30. Moderate scleral injection was noted. Decreased abdominal sounds and a profuse, watery diarrhea were evident. Digital pulses were normal. A fecal sample was positive for strongyles and protozoans. The horse was treated with intravenous fluids, a nonsteroidal antiinflammatory agent, and oxytetracycline. A blood sample for PCR was obtained prior to oxytetracycline therapy. The horse responded to therapy and made an uneventful recovery.
Buffy-coat cells from 8-10 ml of anticoagulated whole blood were obtained by hypotonic lysis and prepared for PCR by proteinase K digestion, as described previously. 6 The nested PCR was performed using two pairs of oligonucleotide primers to amplify a 529-bp fragment of the 16S ribosomal RNA (rRNA) gene of E. risticii. 2 Appropriate positive and negative controls were included in each PCR run. Preliminary verification of identity of the amplified products was provided by Southern hybridization using an internal oligonucleotide probe. 2 For final verification of identity the majority of the 16S rRNA gene from case no. 2 was sequenced, as previously described. 5
Blood buffy-coat cells from both cases of SRC produced amplified 529-bp bands characteristic of E. risticii (Figure 1 ). Southern hybridization and DNA sequencing revealed that the 529-bp bands were indeed 16S rRNA gene fragments from an E. risticii strain. Sequencing of the majority of the 16S rRNA gene (1422 bp) indicated that the SRC agent from the Mt. Shasta horse was most closely related to the Kentucky strain ofE. risticii, with which only 3 nucleotide differences were noted (
Periods indicate conserved positions relative to E. risticii-Illinois, the type strain.
ences were found between the SRC agent and the E. risticii type strain (Illinois), while 6 were noted with the more distantly related Ohio 081 strain (Table 1) . Anorexia, lethargy, diarrhea, and fever are major clinical findings in Potomac horse fever 7-1~ and represent the major clinical signs of SRC as well. Similarly, laminitis is a significant complication in a percentage of cases, s,l~ Interestingly, the gradual spread of SRC farther away from the river as the season progresses is an exact echo of the initial descriptions of Potomac horse fever along the Potomac River. 8 Horses with SRC are indistinguishable clinically from Potomac horse fever cases we have identified in nearby Klamath Falls in southern Oregon, a where the disease is known locally as "ditch fever" from its association with pastures bordering irrigation ditches. The Klamath Falls area is contained within a northern excursion of the Siskiyou/ Shasta ecosystem, a combined upper Sonoran and transition zone that sweeps across portions of southern Oregon but is inhabited by vegetation and wildlife that are clearly Californian. 11 Although the vector of E. risticii remains unidentifiedfl ~ it seems reasonable to suspect that if it is present in Klamath Falls, it may be present in Siskiyou and Shasta counties as well. Available data suggest that the vector is not a tick, but is instead an organism closely associated in some way with river and irrigation water. 2,1~
The nucleotide sequences of 16S rRNA genes are known to vary in an orderly manner throughout the phylogenetic tree; as "molecular chronometers" they exhibit only minor sequence divergence among strains of an individual species. 12 Three major groups of E. risticii based on 16S rRNA gene sequence differences have been described, s These include the type strain (Illinois) and related Ohio isolates; the Kentucky strain; and a more variable strain known as Ohio 081. The genetic differences among these strains are mirrored in their antigenic differences. 5,13 It has been suggested that the degree of divergence shown by the Kentucky and Ohio 081 strains from E. risticii-Illinois may justify their removal from the species risticii and reassignment to separate species status. 5 Thus it is possible that Potomac horse fever (and thus SRC) may be caused by more than a single species of the genus Ehrlichia. Isolation and cultivation of various strains of E. risticii from different areas of the country should improve our understanding of the genetic and antigenic divergence of the organism, and aid in the development of more effective vaccines and serodiagnostic tests.
To summarize, we have provided the first DNA-based evidence that the disease entity known to practitioners in far northern California as the "Shasta River crud" is in fact Potomac horse fever. This part of California may be one of the few areas of the state where conditions necessary for the propagation and transmission of E. risticii and its reservoir hosts/vectors exist; overall the occurrence of Potomac horse fever in California horses appears to be low. 4 Considering that SRC has been recognized in the region for at least 25 years, its clinical discovery appears to predate that of Potomac horse fever itself, which was first reported only in 1979. 6 It is thus interesting to reflect how, but for the vicissitudes of history, the disease caused by E. risticii might just as easily have been named "Shasta horse fever."
Addendum. Since this work was performed we have identified E. risticii by PCR in three additional cases of SRC in Siskiyou County. The first was a febrile 21-yearold Quarter Horse gelding pastured at Horse Creek along the Klamath River, northwest of Mt. Shasta (sample provided by Dr. Paul Miller). The second was a 4-year-old Quarter Horse mare from Montague, along the Shasta River (sample provided by Dr. Bob Cook, Yreka Veterinary Hospital, Yreka, Ca). The third was a febrile 15-yearold Quarter Horse gelding that has lived along the Shasta River for many years (sample provided by Dr. Miller).
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