Abstract
Upon ingestion of adult aquatic insects, horses developed clinical signs of Potomac horse fever, and Neorickettsia risticii was isolated from the blood. 16S rRNA and 51-kDa antigen gene sequences from blood, isolates, and caddis flies fed to the horses were identical, proving oral transmission of N. risticii from caddis flies to horses.
Potomac horse fever (PHF) is caused by Neorickettsia risticii (8). N. risticii DNA has been found in virgulate cercariae from freshwater snails (family Pleuroceridae: Juga yrekaensis) from Northern California (1, 6) and in virgulate xiphidiocercariae isolated from Elimia livescens snails from Ohio (3). N. risticii in trematodes from snails can infect mice by intraperitoneal inoculation (3) and horses by subcutaneous inoculation (5). N. risticii DNA has also been detected in metacercariae from various aquatic insects (2), and recently in Northern California, one horse fed adult caddis flies (family Limnephilidae: Dicosmoecus gilvipes) developed PHF (4). Since, in that study, the N. risticii sequence in the caddis flies fed to the horse was not determined, whether the N. risticii strain in the caddis flies actually infected the horse is unknown. In the present study, we determined (i) a species of snails and adult insects infested with trematodes harboring N. risticii and the N. risticii PCR-positive rates of snail trematodes and larval and adult aquatic insects collected in Pennsylvania, (ii) whether immunocompetent mice can become infected with N. risticii by ingesting adult aquatic insects, (iii) the characteristics of prepatent and ehrlichemia periods and time course of disease development in horses after ingestion of insects, and (iv) the sequences of nearly full-length 51-kDa-antigen genes (12) and 16S rRNA genes of N. risticii from horses and insects fed to the horses.
E. virginica snails, caddis flies, mayflies, and aquatic insect larvae were collected in an area where PHF is endemic, near the Susquehanna River in central Pennsylvania on 14 to 16 August 2000. The nested-PCR (3)-amplified (382-bp) product of the N. risticii 16S rRNA gene was found in 12% (5 of 42 pools from each snail) of sporocysts and cercariae isolated from snails, 11% (4 of 35 pools of two each) of aquatic insect larvae, 20% (2 of 10 pools of two each) of mayflies, and 38% (3 of 8 pools of two each) of caddis flies.
Nine CF-1 mice (4 weeks old) were divided into three groups. The first group was fed aquatic insect larvae, the second was fed adult mayflies (family Polymitarcyidae: Ephoron leukon), and the third was fed adult caddis flies (family Leptoceridae: Oecetis inconspicua) freshly collected on 25 August 2000. By PCR, 0% (0 of 14 pools of two each) of larvae, 6.0% (1 of 15 pools of two each) of mayflies, and 86.7% (13 of 15 pools of two each) caddis flies were N. risticii positive. No mouse showed any signs of illness throughout the 16- to 24-day period. Blood collected at day 6 was positive for one mayfly-fed mouse. One mouse from each group was sacrificed on day 16, 23, or 24, and blood, spleen, and liver DNA samples were collected. One mouse (C3) fed caddis flies and killed on day 24 postfeeding was positive for both the 16S rRNA and 51-kDa-antigen genes by nested PCR. No trematode was found in the mouse intestines. We concluded that immunocompetent mice would serve as a convenient oral-infection model for PHF.
Horse 1 (a 3-year-old gelding) was fed adult mayflies (family Heptageniidae: Leucrocuta minerva) mixed with caddis flies (family Hydropsychidae: Cheumatopsyche campyla and Hydropsyche hageni) on 7 September 2000 (day 0 of feeding) and mayflies alone on 11 September 2000. Horse 2 (a 10-year-old mare) was fed caddis flies (C. campyla and H. hageni) only on 11 September 2000 (day 0 of feeding). Before feeding, both horses were N. risticii negative by PCR and by indirect fluorescence antibody testing. The same batch was used for all feedings, and 0% (0 of 20 pools of two each) of mayflies and 80% of caddis flies (12 of 15 pools of two each) were N. risticii 16S rRNA PCR positive. Horse 1 developed slightly digital pulses and warm feet at day 13, followed by slight depression, anorexia, and decreased borborygmus on day 18, which immediately preceded development of fever and diarrhea on days 19 through 25. Horse 2 developed anorexia, depression, high fever, and decreased borborygmus on day 15 followed by warm feet and digital pulses on day 19. Horse 1 developed leukopenia from days 19 through 22, and horse 2 developed neutropenia from days 11 to 18 and leukopenia from days 11 to 15. Platelet counts decreased starting on day 14 and remained low throughout the study period, but other clinical signs disappeared from horses 1 and 2 by days 27 and 23, respectively, without antibiotic treatment. Horses 1 and 2 seroconverted on days 11 and 7, respectively (Table 1). Since mayflies were PCR negative and time courses of development of clinical signs, PCR, and culture isolation were similar between horses 1 and 2, both horses appear to have acquired N. risticii from caddis flies. Ehrlichemia in horses began 6 to 11 days after ingestion of insects, similar to that in dogs after ingestion of Neorickettsia helminthoeca in Nanophyetus salmincola metacercariae in fish (11). Fewer culture days were required for N. risticii isolation after days 15 and 11 for horses 1 and 2, respectively, suggesting that an increase in organism numbers in the blood preceded the onset of clinical signs (Table 1). Transmission electron microscopy of culture-isolated N. risticii from blood collected on days 15 and 11 postingestion from horses 1 and 2, respectively, revealed ultrastructures clearly resembling that of N. risticii Virginia strain found in experimentally infected horse intestines (10) and in P388D1 cells after isolation from naturally infected horses in Maryland, Ohio, and Kentucky (7, 9).
TABLE 1.
Culture isolation of N. risticii, immunofluorescence assay (IFA) titers, and PCR results for horses 1 and 2 following ingestion of aquatic insects
| Horse | Days postingestion | N. risticii isolation | Days in culture | IFA titer | PCR result |
|---|---|---|---|---|---|
| 1 | 6 | + | 29 | 0 | − |
| 11 | + | 20 | 40 | − | |
| 15 | + | 16 | 40 | + | |
| 19 | + | 12 | 320 | + | |
| 22 | + | 10 | 320 | + | |
| 26 | + | 14 | 2,560 | + | |
| 28 | + | 18 | 2,560 | + | |
| 2 | 2 | − | 34 | 0 | − |
| 7 | − | 29 | 20 | − | |
| 11 | + | 16 | 20 | − | |
| 15 | + | 12 | 160 | + | |
| 18 | + | 8 | 320 | + | |
| 22 | + | 14 | 1,280 | + | |
| 24 | + | 11 | 2,560 | + |
The nearly complete (1,452-bp) 16S rRNA gene sequences found in isolates from horses 1 and 2 and caddis flies fed to the horses and the partial (333-bp) hypervariable region of the 16S rRNA gene sequences obtained from buffy coat DNA of both horses from the earliest PCR-positive dates, from mouse C3 spleen tissue, and from adult insects (both mayflies and caddis flies) fed to mice were identical (Table 2). Since the 16S rRNA gene sequence is highly conserved, we also sequenced the nearly complete 51-kDa-antigen gene (1,449 bp). The almost complete amino acid sequences of a 51-kDa protein (483 of 558 amino acids [aa] [87.0% of the total aa]) in culture isolates from horses 1 and 2 and the caddis flies fed to the horses and the partial sequence (171 aa) in blood from horses 1 and 2 and in mouse C3 spleen tissue were identical, proving the transmission of N. risticii from adult caddis flies to the horses and a mouse (Table 3). Comparison of amino acid sequences of the 51-kDa antigen suggested a geographic segregation of N. risticii strains. Sequences from N. risticii DNA originating in the eastern United States (Ohio, Maryland, Kentucky, and Pennsylvania) were least identical to sequences originating from California (Table 4) (3). The 16S rRNA gene sequence and 51-kDa-antigen amino acid sequences of the Ohio 081 isolate remain quite divergent from both those in eastern and western N. risticii groups (Tables 3 and 4).
TABLE 2.
Nucleotide differences between 16S rRNA genes of N. risticii from selected sources
| Source | Nucleotide at positiona:
|
||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 92 | 131 | 168 | 186 | 242 | 309 | 382 | 395 | 436 | 619 | 956 | 971 | 1043 | 1221 | 1226 | 1231 | 1246 | |
| Type strain | T | G | A | A | G | A | C | G | A | G | T | G | G | C | G | T | G |
| Horse 1 isolateb | • | • | • | • | • | • | • | • | • | • | • | • | — | • | • | • | • |
| Horse 2 isolatec | • | • | • | • | • | • | • | • | • | • | • | • | — | • | • | • | • |
| Caddis flyd | • | • | • | • | • | • | • | • | • | • | • | • | — | • | • | • | • |
| Caddis flye | • | • | • | • | • | • | • | • | • | / | / | / | / | / | / | / | / |
| Horse 1 bloodf | • | • | • | • | • | • | • | • | • | / | / | / | / | / | / | / | / |
| Horse 2 bloodg | • | • | • | • | • | • | • | • | • | / | / | / | / | / | / | / | / |
| Mouse C3h | • | • | • | • | • | • | • | • | • | / | / | / | / | / | / | / | / |
| Mayflyi | • | • | • | • | • | • | • | • | • | / | / | / | / | / | / | / | / |
| Eclipsej | • | • | • | • | • | • | T | • | • | • | • | • | • | • | A | • | • |
| S22k | • | • | • | • | • | • | • | • | • | • | • | • | • | • | • | • | • |
| S6k | • | A | • | • | • | • | • | • | • | • | • | • | • | • | • | • | • |
| SHSN-1l | • | • | • | • | • | G | • | • | • | • | C | • | • | T | • | T | A |
| Ohio 081m | C | • | • | • | • | • | • | • | • | T | C | A | • | T | • | T | A |
Bullets represent conserved nucleotides (relative to the sequence of the type strain; GenBank no. M21290). Slashes indicate that the 16S rRNA sequence was not completely determined. Dashes indicate gaps in the sequence.
Culture isolate from horse 1, 15 days after feeding with caddis flies (C. campyla and H. hageni) (GenBank no. AF380257).
Culture isolate from horse 2, 11 days after feeding with caddis flies (GenBank no. AF380258).
Caddis flies (C. campyla and H. hageni) collected in central Pennsylvania and fed to horses (GenBank no. AF380264).
Caddis flies (O. inconspicua) collected in central Pennsylvania and fed to mice (GenBank no. AF380262).
Buffy coat from horse 1, 11 days after caddis fly feeding (GenBank no. AF380259).
Buffy coat from horse 2, 15 days after caddis fly feeding (GenBank no. AF380260).
Spleen of mouse fed infected caddis flies (GenBank no. AF380261).
Mayflies (E. leukon) collected in central Pennsylvania and fed to mice (GenBank no. AF380263).
Horse in Elizabethville, Pa. (GenBank no. AF036653).
Virgulate trematode pool isolated from E. livescens in Warsaw, Ohio (GenBank no. AY005439).
Juga sp. in Weed, Calif. (GenBank no. AF037210).
Isolate from a horse in Findlay, Ohio (13).
TABLE 3.
Amino acid differences of 51-kDa antigens among N. risticii sources
| Source | Amino acid at indicated positiona
|
||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 95 | 108 | 114 | 128 | 135 | 141 | 142 | 143 | 144 | 145 | 146 | 147 | 148 | 149 | 150 | |
| 90-12 strainb | R | S | K | A | S | T | S | E | A | N | S | N | S | V | N |
| Horse 1c | • | • | • | • | • | • | • | • | • | • | • | • | • | • | • |
| Horse 2d | • | • | • | • | • | • | • | • | • | • | • | • | • | • | • |
| Caddis flye | / | / | / | / | / | / | / | / | / | / | / | / | • | • | • |
| Mouse C3f | / | / | / | / | / | / | / | / | / | / | / | / | • | • | • |
| S21g | / | / | / | / | / | / | / | / | • | • | • | • | • | • | • |
| Eclipseh | / | / | / | / | / | • | • | • | • | • | • | • | • | • | • |
| Jugai | / | / | / | / | / | • | • | G | S | G | • | • | T | A | S |
| SHSN-1j | / | / | / | / | / | • | • | G | S | G | • | • | T | A | S |
| Ohio 081k | K | R | E | T | A | A | N | — | — | — | — | — | • | T | N |
| 151 | 154 | 163 | 185 | 187 | 199 | 204 | 226 | 230 | 232 | 236 | 239 | 247 | 250 | 262 | |
| 90-12 strainb | N | A | D | G | F | R | A | D | Q | I | V | I | N | S | L |
| Horse 1c | • | • | • | • | • | • | • | • | • | • | • | • | • | • | • |
| Horse 2d | • | • | • | • | • | • | • | • | • | • | • | • | • | • | • |
| Caddis flye | • | • | • | • | • | • | • | • | • | • | • | • | • | • | • |
| Mouse C3f | • | • | • | • | • | • | • | • | • | • | • | • | • | • | • |
| S21g | • | • | • | • | • | • | • | • | • | • | • | • | • | • | • |
| Eclipseh | • | • | • | • | L | • | • | • | • | • | • | • | • | • | • |
| Jugai | S | T | • | • | • | • | • | • | E | • | • | • | • | • | • |
| SHSN-1j | S | T | • | • | • | • | • | • | E | • | • | • | • | • | • |
| Ohio 081k | • | • | N | S | • | K | S | E | K | L | A | V | E | N | I |
| 263 | 266 | 267 | 269 | 270 | 271 | 272 | 273 | 274 | 274l | 275 | 276 | 277 | 282 | 284 | |
| 90-12 strainb | G | H | S | G | T | G | S | S | S | — | Q | S | K | V | P |
| Horse 1c | • | • | • | • | • | • | • | • | • | — | • | • | • | • | R |
| Horse 2d | • | • | • | • | • | • | • | • | • | — | • | • | • | • | R |
| Caddis flye | • | • | • | • | • | • | • | • | • | — | • | • | • | • | R |
| Mouse C3f | • | • | • | • | • | • | • | • | • | — | • | • | • | • | R |
| S21g | • | • | • | • | • | • | • | • | • | — | • | • | • | • | R |
| Eclipseh | • | • | • | • | • | • | • | • | • | — | • | • | • | • | R |
| Jugai | • | • | N | • | • | S | G | N | N | — | • | • | • | • | H |
| SHSN-1j | • | • | N | • | • | S | G | N | N | — | • | • | • | • | H |
| Ohio 081k | N | R | T | N | — | — | G | • | T | T | T | C | G | A | H |
| 286 | 290 | 291 | 294 | 295 | 312 | 319 | 331 | 333 | 336 | 340 | 347 | 358 | |||
| 90-12 strainb | S | A | D | S | P | F | S | I | V | G | K | P | P | ||
| Horse 1c | F | • | • | T | • | L | T | • | • | • | • | A | A | ||
| Horse 2d | F | • | • | T | • | L | T | • | • | • | • | A | A | ||
| Caddis flye | F | • | • | T | • | L | T | / | / | / | / | / | / | ||
| Mouse C3f | F | • | • | T | • | L | T | / | / | / | / | / | / | ||
| S21g | F | • | • | T | • | L | / | / | / | / | / | / | / | ||
| Eclipseh | F | • | • | T | • | L | T | / | / | / | / | / | / | ||
| Jugai | F | • | N | T | • | L | • | / | / | / | / | / | / | ||
| SHSN-1j | F | • | N | T | • | L | • | / | / | / | / | / | / | ||
| Ohio 081k | F | T | N | T | Q | L | T | V | L | A | Q | G | / | ||
Bullets represent conserved amino acids (relative to the sequence of strain 90- 12). Slashes indicate that the amino acid sequence was not determined. Dashes indicate gaps in the sequence. Sequences available from GenBank were used for comparison.
Isolate from a horse in Maryland (GenBank no. U85784).
Culture isolate from horse 1 (GenBank no. AF380265).
Culture isolate from horse 2 (GenBank no. AF380266).
Caddis flies collected in western Pennsylvania and fed to horses (GenBank no. AF380270).
Spleen of mouse fed infected caddis flies (GenBank no. AF380269).
Virgulate trematode pool isolated from E. livescens in Warsaw, Ohio (GenBank no. AY005440).
Horse in Elizabethville, Pa. (GenBank no. AF036673).
Secretions from Juga spp. in Weed, Calif. (GenBank no. AF036674).
Juga sp. in Weed, Calif. (GenBank no. AF037215).
Isolate from a horse in Findlay, Ohio (GenBank no. AY005443).
This position is absent in strain 90-12.
TABLE 4.
Amino acid sequence identities and evolutionary distances between 51-kDa antigen sequences from selected N. risticii sources
| Source | % Amino acid sequence identity or evolutionary distancea:
|
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| 90-12 | Horse 1 | Horse 2 | Caddis fly | Mouse C3 | S21 | Eclipse | Juga | SHSN-1 | Ohio 081 | |
| 90-12b | 0.008 | 0.008 | 0.008 | 0.008 | 0.008 | 0.010 | 0.089 | 0.089 | 0.217 | |
| Horse 1c | 99.2 | 0.000 | 0.000 | 0.000 | 0.000 | 0.002 | 0.082 | 0.082 | 0.209 | |
| Horse 2d | 99.2 | 100.0 | 0.000 | 0.000 | 0.000 | 0.002 | 0.082 | 0.082 | 0.209 | |
| Caddis flye | 99.2 | 100.0 | 100.0 | 0.000 | 0.000 | 0.002 | 0.082 | 0.082 | 0.209 | |
| Mouse C3f | 99.2 | 100.0 | 100.0 | 100.0 | 0.000 | 0.002 | 0.082 | 0.082 | 0.209 | |
| S21g | 99.2 | 100.0 | 100.0 | 100.0 | 100.0 | 0.002 | 0.082 | 0.082 | 0.209 | |
| Eclipseh | 99.0 | 99.8 | 99.8 | 99.8 | 99.8 | 99.8 | 0.084 | 0.084 | 0.212 | |
| Jugai | 91.0 | 91.6 | 91.6 | 91.6 | 91.6 | 91.6 | 91.4 | 0.000 | 0.224 | |
| SHSN-1j | 91.0 | 91.6 | 91.6 | 91.6 | 91.6 | 91.6 | 91.4 | 100.0 | 0.224 | |
| Ohio 081k | 79.1 | 79.7 | 79.7 | 79.7 | 79.7 | 79.7 | 77.5 | 79.3 | 77.3 | |
Values in the upper right portion are evolutionary distances; values in the lower left portion are percent amino acid sequence identities. The values were calculated by comparison of a total of 169 or 170 amino acids of the smallest sequence from the alignment.
Isolate from a horse in Maryland (GenBank no. U85784).
Culture isolate from horse 1 (GenBank no. AF380265).
Culture isolate from horse 2 (GenBank no. AF380266).
Caddis flies collected in central Pennsylvania and fed to horses (GenBank no. AF380270).
Spleen of mouse fed infected caddis flies (GenBank no. AF380269).
Virgulate trematode pool isolated from E. livescens in Warsaw, Ohio (GenBank no. AY005440).
Horse in Elizabethville, Pa. (GenBank no. AF036673).
Secretions from Juga spp. in Weed, Calif. (GenBank no. AF036674).
Juga sp. in Weed, Calif. (GenBank no. AF037215).
Isolate from a horse in Findlay, Ohio (GenBank no. AY005443).
Acknowledgments
This work was supported by National Research Initiative Competitive Grant 99-35204-8521 from the U.S. Department of Agriculture and The Ohio State University Equine Research Fund.
We thank Manuel Kanter for his help in sample collection, preparation, and PCR. We thank Brian J. Armitage and G. Thomas Waters, Museum of Biological Diversity at the Ohio State University, for identification of the caddis flies and snails, respectively. We thank Michael D. Hubbard, Entomology Center, Florida A&M University, for identification of the mayflies. For instruction on collecting aquatic insect larvae, we thank Eric J. Wetzel at Wabash College, Crawfordsville, Indiana. We thank Richard Berry for his advice on insect collection techniques. We also thank Clifton M. Monahan for his discussion on trematode biology. We thank J. Scott Martin for his assistance in our horse transmission study. For PCR of horse blood samples, we thank Mingqun Lin, Department of Veterinary Biosciences at the Ohio State University.
REFERENCES
- 1.Barlough, J. E., G. H. Reubel, J. E. Madigan, L. K. Vredevoe, P. E., Miller, and Y. Rikihisa. 1998. Detection of Ehrlichia risticii, the agent of Potomac horse fever, in freshwater stream snails (Pleuroceridae: Juga spp.) from northern California. Appl. Environ. Microbiol. 64:2888-2893. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Chae, J.-S., N. Pusterla, E. Johnson, E. DeRock, S. P. Lawler, and J. E. Madigan. 2000. Infection of aquatic insects with trematode metacercariae carrying Ehrlichia risticii, the cause of Potomac horse fever. J. Med. Entomol. 37:619-625. [DOI] [PubMed] [Google Scholar]
- 3.Kanter, M., J. Mott, N. Ohashi, B. Fried, S. Reed, Y. Lin, and Y. Rikihisa. 2000. Analysis of 16S rRNA and 51-kilodalton antigen gene and transmission in mice of Ehrlichia risticii in virgulate trematodes from Elimia livescens snails in Ohio. J. Clin. Microbiol. 38:3349-3358. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Madigan, J. E., N. Pusterla, E. Johnson, J.-S. Chae, J. B. Pusterla, E. DeRock, and S. P. Lawler. 2000. Transmission of Ehrlichia risticii, the agent of Potomac horse fever, using naturally infected aquatic insects and helminth vectors: preliminary report. Equine Vet. J. 32:275-279. [DOI] [PubMed] [Google Scholar]
- 5.Pusterla, N., J. E. Madigan, J. Chae, E. DeRock, E. Johnson, and J. B. Pusterla. 2000. Helminthic transmission and isolation of Ehrlichia risticii, the causative agent of Potomac horse fever, by using trematode stages from freshwater stream snails. J. Clin. Microbiol. 38:1293-1297. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Reubel, G. H., J. E. Barlough, and J. E. Madigan. 1998. Production and characterization of Ehrlichia risticii, the agent of Potomac horse fever, from snails (Pleuroceridae: Juga spp.) in aquarium culture and genetic comparison to equine strains. J. Clin. Microbiol. 36:1501-1511. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Rikihisa, Y. 1991. The tribe Ehrlichieae and ehrlichial diseases. Clin. Microbiol. Rev. 4:286-308. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Rikihisa, Y. 1998. Rickettsial diseases in horses, p. 112-123. In W. Bayly and S. M. Reed (ed.), Equine internal medicine. The W.B. Saunders Co., Philadelphia, Pa.
- 9.Rikihisa, Y., W. Chaichanasiriwithaya, S. M. Reed, and S. Yamamoto. 1995. Distinct antigenic differences of new Ehrlichia risticii isolates, p. 211-215. In Proceedings of the 7th International Conference on Equine Infectious Diseases, 1994, Tokyo, Japan. R&W Publishing, Newmarket, United Kingdom.
- 10.Rikihisa, Y., B. D. Perry, and D. O. Cordes. 1985. Ultrastructural study of rickettsial organisms in the large colon of ponies experimentally infected with Potomac horse fever. Infect. Immun. 49:505-512. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Rikihisa, Y., H. Stills, and G. Zimmerman. 1991. Isolation and continuous culture of Neorickettsia helminthoeca in a macrophage cell line. J. Clin. Microbiol. 29:1928-1933. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Vemulapalli, R., B. Biswas, and S. K. Dutta. 1998. Cloning and molecular analysis of genes encoding two immunodominant antigens of Ehrlichia risticii. Microb. Pathog. 24:361-372. [DOI] [PubMed] [Google Scholar]
- 13.Wen, B., Y. Rikihisa, P. A. Fuerst, and W. Chaichanasiriwithaya. 1995. Diversity of 16S rRNA genes of new Ehrlichia strains isolated from horses with clinical signs of Potomac horse fever. Int. J. Syst. Bacteriol. 45:315-318. [DOI] [PubMed] [Google Scholar]