Detection of serum antibodies against Ehrlichia risticii in Potomac horse fever by enzyme-linked immunosorbent assay

An indirect enzyme-linked immunosorbent assay (ELISA) was developed which was specific and sensitive in detecting antibodies to Ehrlichia risticii in Potomac horse fever (PHF). The ELISA antibody titers were correlated with the indirect fluorescent antibody (IFA) titers. E. risticii propagated in human histiocyte culture was purified on renografin gradient and the band of the organisms at a density of 1.182 g/ml was used as antigen. ELISA antibody titers were determined through computer assisted analysis, the observed antibody titers were derived by serial serum dilutions and using a resultant standard curve the predicted antibody titers were obtained from a single serum dilution. The standard curve had a correlation coefficient of 0.8975. The observed and predicted antibody titers were in good agreement, as the respective titers fell within a two-fold range. There was a good correlation between ELISA and IFA test results, but the ELISA titers were several times higher. In experimental infections of horses produced with the infected equine whole blood and the Ehrlichia infected macrophage culture, the antibodies were first detected in two weeks and one week postinoculation (PI), respectively. In both cases the titers reached a peak in about 4 weeks PI with a mean titer of 1:16558 and 1:4030, respectively. The antibody titers of the convalescent sera of field cases of PHF were comparatively lower than the experimentally infected horses.     

Infection rate of Ehrlichia risticii, the agent of Potomac horse fever, in freshwater stream snails (Juga yrekaensis) from northern California

Juga yrekaensis freshwater snails were tested for trematode stages and for Ehrlichia risticii DNA using a nested PCR assay. Snails were collected monthly from two Potomac horse fever (PHF) endemic locations in northern California (Montague and Weed).The trematode infection rate varied between 40 and 93.3% in large snails (shell size >15mm) and between 0 and 13.3% in small snails (<15mm). The highest trematode infection rate for large and small snails was recorded in September and the lowest infection rate for large snails was recorded in June (Weed) and October (Montague). The E. risticii PCR infection rate among small snails from both sites was similar and varied monthly between 0 and 3.3%. The PCR infection rate for large snails from Weed was high in May (20.0%) and decreased progressively until November (10.0%). The PCR infection rate for large snails from Montague was 5.0% in May, 26.3% in August and 16. 7% in October. PCR-positive snails were always related to the microscopic detection of trematode stages (virgulate cercariae).This study provides evidence that J. yrekaensis are infected with trematode cercariae that harbor E. risticii. The number of snails harboring trematode stages and the number of PCR positive snails varied with the size of the snails, the month of collection, and the geographic origin.     

Susceptibility of dogs to infection with Ehrlichia risticii, causative agent of equine monocytic ehrlichiosis (Potomac horse fever)

Adult dogs 1 to 5 were inoculated IV and/or SC with 3, 5, or 6 ml of a suspension containing 1.2 x 10(4) Ehrlichia risticii-infected cells (derived from primary canine monocyte cell cultures)/ml. Dogs 6 to 8 were inoculated IV and/or SC with 3 or 6 ml of 1.2 x 10(5) organism-free cultured canine monocytes/ml. Ehrlichia risticii was isolated in cultures from inoculated dogs 3, 4, and 5 on postinoculation days (PID) 10 to 16, but not from dogs 6 to 8. Dogs inoculated with E risticii seroconverted between PID 6 and 12. Clinical signs of illness were not observed in these 5 E risticii-inoculated dogs. A pony, inoculated with E risticii isolated from inoculated dog 5, developed clinical signs of equine monocytic ehrlichiosis, including fever, anorexia, depression, and diarrhea, and E risticii was isolated from the pony's blood. This E risticii isolate was then inoculated into susceptible dog 9, and E risticii was repeatedly isolated from dog 9 during PID 6 to 17. Dogs were susceptible to infection with E risticii and may serve as a reservoir of the organism in the field.     

Transmission of Ehrlichia risticii, the agent of Potomac horse fever, using naturally infected aquatic insects and helminth vectors: preliminary report

Ehrlichia risticii, the agent of Potomac horse fever (PHF), has been recently detected in trematode stages found in snail secretions and in aquatic insects. Based on these findings, horses could conceivably be exposed to E. risticii by skin penetration with infected cercariae, by ingestion of infected cercariae in water or via metacercariae in a second intermediate host, such as an aquatic insect. In order to test this hypothesis, horses were challenged with infectious snail secretions and aquatic insects collected from a PHF endemic region in northern California. Two horses stood with their front feet in water harbouring E. risticii-infected cercariae, 2 horses drank water harbouring E. risticii-infected cercariae, and 6 horses were fed pools of different aquatic insects harbouring E. risticii-infected metacercariae. In this preliminary study, only the one horse infected orally with mature caddisflies (Dicosmoecus gilvipes) developed the clinical and haematological disease syndrome of PHF. The agent was isolated from the blood of the infected horse in a continuous cell line and identified as E. risticii by characterisation of the 16S rRNA gene. Therefore, E. risticii is maintained in nature in a complex aquatic ecosystem and transmission to horses can occur through accidental ingestion of insects such as caddisflies containing infected metacercariae. At present, the small number of horses used in this study does not exclude other insects and free trematode stages as potential sources of infection.     

Sarcocystis myositis and vitamin E deficiency in a Gypsy Vanner stallion suspected of having equine motor neuron disease

A 3-year-old Gypsy Vanner stallion was presented for evaluation of intermittent recumbency, muscle fasciculations, weakness, low head carriage, shifting of weight between the hindlimbs and an elevated tail head. History, physical examination and serum alpha tocopherol concentrations were suggestive of vitamin E deficiency and equine motor neuron disease (EMND). Sacrocaudalis dorsalis medialis muscle biopsy identified myositis secondary to sarcocystosis. Treatment with alpha tocopherol, ponazuril and sulfadiazine/pyrimethamine resulted in significant improvement in muscle weakness and body condition with resolution of sarcocystosis and inflammation on repeat muscle biopsy. This case illustrates the importance of muscle biopsy in horses with neuromuscular disease as concurrent diseases may be present that require specific treatment for a positive outcome.     

Enterocolitis caused by Ehrlichia sp. in the horse (Potomac horse fever)

Potomac horse fever was reproduced in 15 ponies by transfusion of whole blood originally from two natural cases and subsequently from ponies infected by the transfusions. Incubation periods varied from 9 to 15 days. Affected ponies developed varying degrees of fever, diarrhea, anorexia, depression, and leukopenia. Eleven affected ponies were killed, three died in the acute phase of the disease, and one did not show clinical signs. The most consistent post-mortem findings were fluid contents in the cecum and large colon, and areas of hyperemia (of inconstant degree and distribution) in mucosae of both small and large intestines. Multifocal areas of necrosis occurred in mucous membranes. Ehrlichial organisms were most common in the cytoplasm of epithelial cells, macrophages, and mast cells of the large colon.     

Potomac horse fever in Ontario: Clinical,geographic, and diagnostic aspects

Clinical findings, geographic locations, laboratory diagnoses, and culture isolation of Neorickettsia spp. in Potomac horse fever (PHF) cases diagnosed in Ontario between 2015 and 2019 are described. Forty-six confirmed PHF cases occurred from late June to early September. Of 41 horses admitted to the Ontario Veterinary College, 28 (68%) survived and 13 (32%) were euthanized due to poor prognosis or financial constraints. Most cases were in southern Ontario along the Canada-USA border. Blood and fecal samples from 43 suspect PHF cases were submitted to 2 laboratories for polymerase chain reaction (PCR) testing for Neorickettsia risticii. Agreement between both laboratories for detection of N. risticii DNA was excellent for feces [κ = 0.932, 95% confidence interval (CI): 0.80 to 1], and fair for blood samples (κ = 0.494, 95% CI: 0.13 to 0.85). Neorickettia spp. were isolated from 16 of 41 (39%) blood samples. DNA analysis confirmed 14 isolates were N. risticii and 2 were N. findlayensis, a novel species of Neorickettsia recently demonstrated to cause PHF.     

Two cases of Neorickettsia (Ehrlichia) risticii infection in horses from Nova Scotia

Two horses from Nova Scotia were diagnosed with Potomac horse fever (PHF). Polymerase chain reaction analysis was performed on formalin-fixed colon tissue or whole blood to show the presence of Neorickettsia risticii DNA, the causative agent of PHF. These are the first reported cases of PHF in the Maritime Provinces.     

Diagnosis of equine monocytic ehrlichiosis (Potomac horse fever) by indirect immunofluorescence

The recent establishment of a system for the continuous in vitro propagation of Ehrlichia risticii, the causative agent of equine monocytic ehrlichiosis (EME; synonym, Potomac horse fever), has facilitated the development of an indirect fluorescent antibody test for the diagnosis of this disease under laboratory and field conditions. The field diagnostic application of the test has aided in the recognition of the disease in 16 states of the United States and in 1 province of Canada. A limited epidemiologic study conducted between January and September 1985, in an area where the disease is known to be enzootic, revealed that conversion from seronegative to seropositive status is not always accompanied by clinical manifestations of the disease. Confirmatory findings in experimentally inoculated horses suggest the existence of clinically undetectable infections.     

Epidemiology of Potomac horse fever: an investigation into the possible role of non-equine mammals

A serological study of antibodies to Ehrlichia risticii was carried out on 10 species of wild and domestic mammals found on or near 21 horse farms in an area of the USA in which Potomac horse fever is endemic. No antibodies were found in 133 peridomestic rodents (Norway rats and house mice), nor in 108 wild rodents (white-footed mice and meadow voles) captured on farms. Three of the six domestic animal species examined, cats, pigs and a goat, showed serological evidence of exposure to E risticii. Seropositive animals were detected on three of the 21 premises. The eight seropositive cats (of 48 cats tested) were on two farms, and the three seropositive pigs (of 14 tested) were all on one farm which lay some 3 km from where the one seropositive goat (of three tested) was found. None of the 79 dogs, 75 cattle and seven sheep tested had antibodies to E risticii. The significance of these findings is discussed in the light of current understanding of the transmission of Potomac horse fever and of the epidemiology of other related ehrlichial diseases.     

Loss of absorptive capacity for sodium and chloride in the colon causes diarrhoea in Potomac horse fever

Ehrlichia risticii, an obligate intracellular bacterium in the family Rickettsiaceae, causes Potomac horse fever which is often associated with severe watery diarrhoea. The mechanism of the diarrhoea is unknown. The aim of this study was to determine whether sodium and chloride transport, morphology and cyclic adenosine 3', 5'-monophosphate (cyclic AMP) content of colonic mucosa was altered in E risticii-infected horses. Mucosa-submucosa sheets from the large and small colon of nine infected and seven to nine uninfected horses were set up in Ussing chambers for measurement of short-circuit current and transepithelial 22Na and 36Cl fluxes. Uninfected tissues absorbed both sodium and chloride whereas absorption of sodium and chloride was abolished in infected tissues. Bethanechol and histamine evoked a concentration-dependent increase in short-circuit current in both groups, but the responses were attenuated at all concentrations in infected horses. Slight focal degeneration of colonic epithelial cells and loss of microvilli from glandular epithelial cells occurred in infected horses. There was a significant increase in cyclic AMP content in colonic mucosa of infected animals. The results suggest that E risticii infection induces focal microscopic degeneration of epithelial cells and an increase in intracellular cyclic AMP in colonic mucosa. These alterations are associated with malabsorption of sodium and chloride and could cause diarrhoea.