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

Abstract

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.

Case 1

A 6-year-old, 455 kg, quarter horse gelding from Dartmouth, Nova Scotia, was presented for fever of unknown origin. He had a 5-day history of depression, anorexia, decreased fecal output, and elevated body temperature (38.8°C to 40.5°C). The referring veterinarian had treated him with phenylbutazone, flunixin meglumine, and procaine penicillin G, with minimal response. He had been moved recently from Amherst, Nova Scotia, to Dartmouth, Nova Scotia, but had never traveled outside the Maritime Provinces. In both locations, he was kept on pastures in close proximity to water.

Upon initial physical examination at the Atlantic Veterinary College teaching hospital, the gelding was sweating, anxious, estimated to be 5% dehydrated, tachycardic (68 beats/min) and febrile (39.1°C). His mucous membranes were injected and capillary refill time was 2 s. He had decreased gastrointestinal sounds on abdominal auscultation.

Venous blood was collected at admission for a complete blood cell (CBC) count and serum biochemical profile. Results showed a normal hematocrit (0.42 L/L; reference range, 0.32 to 0.52 L/L); marked leukopenia (1.4 × 10⁹ cells/L; reference range, 5.5 to 12.5 × 10⁹ cells/L), characterized by a marked neutropenia (0.08 × 10⁹ cells/L) with severe degenerative left shift (0.22 × 10⁹ cells/L bands; reference range, 0.0 to 0.1 × 10⁹/L) and marked toxic change; mild lymphopenia (1.05 × 10⁹ cells/L; reference range, 1.5 to 5.5 × 10⁹ cells/L); mild azotemia (blood urea nitrogen [BUN] 7.9 mmol/L; reference range, 3.5 to 7.0 mmol/L); mild elevations in muscle enzymes (creatine kinase 1339; reference range, 0 to 500 U/L); and moderate hypoproteinemia (48 g/L; reference range, 60 to 77 g/L) and hypoalbuminemia (20 g/L; reference range, 25 to 36 g/L). Initial treatment included lactated Ringer’s solution, IV, administered at a rate of 4 L/h for the first 6 h to correct dehydration, and then reduced to 2 L/h to maintain hydration, since his water intake was decreased. Treatment with sodium penicillin (Sodium penicillin; Novopharm, Toronto, Ontario), 22 000 IU/kg body weight (BW), IV, q6h; gentamicin (Gentocin; Schering-Plough, Pointe Claire, Quebec), 6.6 mg/kg BW, IV, q24h; and flunixin meglumine (Cronyxin; Vetrepharm, Belleville, Ontario), 1.1 mg/kg BW, IV, q12h was initiated.

Results from thoracic radiographs, and cardiac and abdominal ultrasonographs were unremarkable. An abdominocentesis was attempted, but it was not diagnostic due to blood contamination. Blood for culture and a fecal sample were collected at admission. The feces were cultured for the presence of Salmonella spp. Screening for Clostridium difficile and Clostridium perfringens toxins was not performed. During his 2nd day of hospitalization, the gelding developed profuse watery diarrhea and remained mildly febrile (38.8°C), severely depressed, and anorexic.

On the 3rd day of hospitalization, the gelding’s diarrhea worsened. He developed severe clinical signs of acute laminitis, bounding digital pulses, and a reluctancy to move or lift his feet. He progressed quickly to total recumbency. Abaxial sesamoid nerve blocks were performed on both front limbs, this made him more comfortable and he stood for 1 h before becoming recumbent again. Due to the signs of acute laminitis and the worsening of the diarrhea, the client elected to euthanize the gelding.

Postmortem examination revealed a diffuse, severe, erosive, and ulcerative enterocolitis with marked villous blunting and fusion. Secondary lesions due to bacteremia or endotoxemia were also seen — specifically, renal infarcts, interstitial nephritis, periportal hepatitis, and adrenalitis. No Salmonella spp. or other significant aerobic pathogens were isolated from cultures of the gastrointestinal tract or mesenteric lymph nodes. The blood culture showed no bacterial growth and the fecal cultures were negative for Salmonella spp.

The patient’s clinical signs were also consistent with those of Potomac horse fever (PHF); specifically a period of fever, anorexia, leukopenia, and depression, followed by an acute enterocolitis and laminitis (1). The possibility of an infection with Neorickettsia risticii, the causative agent of Potomac horse fever, was further investigated. Since the horse was euthanized, acute and convalescent titers could not be obtained. Formalin-fixed colon tissue was submitted to the University of California Davis Ehrlichial Research Laboratory for DNA extraction and polymerase chain reaction (PCR) analysis for N. risticii DNA (2). Results were positive for N. risticii.

Case 2

A 1-year-old quarter horse gelding was presented for fever (up to 40.5°C), depression of 2 d duration, and diarrhea, which began 1 d prior to presentation. The referring veterinarian had treated the gelding with flunixin meglumine and IV fluids before referring him to the Atlantic Veterinary College Teaching Hospital. He had been born and raised on the same farm as case 1 in Dartmouth, Nova Scotia; he had never traveled outside the Maritime Provinces.

Upon arrival at the AVC teaching hospital the yearling was depressed, and febrile (39.6°C). He exhibited signs of endotoxic shock, including tachycardia (72 beats/min), injected dark mucous membranes, and cold extremities. He also had watery fetid diarrhea. Venous blood was collected at admission for a CBC count, serum biochemical analysis, and blood gas analysis. Results showed an increased hematocrit (0.46 L/L); a normal white blood cell count (6.3 × 10⁹ cells/L), characterized by a regenerative left shift (2.77 × 10⁹ neutrophils/L, 1.32 × 10⁹ bands/L) with marked toxic changes; markedly elevated fibrinogen (8 g/L; reference range, < 5.0 g/L); plasma protein within normal limits (61 g/L); marked hyponatremia (Na+ 118.4 mmol/L; reference range, 135 to 148 mmol/L); hypochloremia (Cl- 93.2 mmol/L; reference range, 98 to 110 mmol/L); and marked azotemia (BUN 15.4 mmol/L). Metabolic acidosis (pH 7.24, HCO₃ 15.3 mmol/L; reference range, pH 7.32 to 7.44) was also present.

Initial treatment included lactated Ringer’s solution, IV, administered at a rate of 3 L/h after administering the first 6 L as a bolus. Sodium bicarbonate, 50 meq/L, and potassium chloride, 20 meq/L, were added to the fluids. After initial rehydration, the gelding was administered flunixin meglumine (Cronyxin; Vetrepharm), 0.5 mg/kg BW, IV, q12h, and oxytetracycline (Tetrajet LP; Bimeda — MTC Animal Health, Cambridge, Ontario), 6.6 mg/kg BW, IV, q12h, for treatment of possible PHF, since he came from the same farm as case 1. Whole blood was submitted for the PCR test for N. risticii, and serum was submitted for indirect fluorescent antibody (IFA) test for antibody for N. risticii. The yearling’s metabolic status continued to deteriorate, and the owners elected to have him euthanized 17 h after presentation. Both the IFA test and the PCR test were positive for N. risticii.

Neoricketsia risticii, formerly named Ehrlichia risticii, is a rickettsial organism that has an affinity for monocytes and intestinal epithelial cells (3). Potomac horse fever was first reported along the Potomac River in 1983 (1). Since then, it has been diagnosed in many parts of the United States and in Ontario and Alberta (4–7). To the author’s knowledge, this is the 1st documented case of PHF in the Maritime Provinces and the first to report the use of PCR on formalin-fixed tissue to diagnose PHF.

It is important for local veterinarians to know that PHF exists in the Maritimes, so that they may consider it as a differential diagnosis for fever, anorexia, depression, and diarrhea. Tentative diagnosis of PHF usually relies on clinical signs, seasonality, and environment. Severity of clinical signs can range from mild depression and fever to the presentation reported here with severe colitis and laminitis. Initial signs of PHF are depression, anorexia, and fever (38.9°C to 41.7°C); intestinal motility is usually decreased at this stage. Most infected horses will develop diarrhea within 48 h of onset of clinical signs (1,3). Laminitis occurs in 20% to 30% of cases, usually within 3 d of the onset of diarrhea (1,8). Hematologic changes in the early stage of the disease can vary from normal to leukopenic, characterized by a neutropenia and lymphopenia. A marked leukocytosis is a common finding later in the course of disease (8).

Potomac horse fever is seen in endemic areas from spring through the fall. The disease usually affects horses that are housed near creeks or rivers (6). Identification of antibodies to N. risticii in the serum has been used in the past to diagnose PHF; however, there is a high rate of false positives with the IFA test if a single sample is used, because the antibody response to the organism is highly variable (7). A single IFA titer can not differentiate between an active infection and past exposure or vaccination. Thus paired serum samples, including a convalescent sample collected at least 1 wk post clinical signs is required to support a diagnosis of PHF. This makes diagnosis via IFA especially difficult in cases of mortality. A real-time PCR test has been developed for N. risticii that detects the presence of N. risticii DNA in the blood or feces in infected horses (2). The PCR test is as sensitive as cell culture and more specific than IFA (9). The PCR test can be performed on feces or on blood mononuclear cells and can detect the organism at the onset of clinical signs (9–11). In the first case, DNA was extracted from formalin-fixed colon tissue to enable a postmortem diagnosis to be made, the 1st time that the use of this technique to make a postmortem diagnosis has been reported. In the 2nd case, whole blood was used for the PCR, which is preferable to achieve faster results.

Early diagnosis and treatment of PHF are important, because treatment with oxytetracycline, IV, can be successful, especially if started early in the course of the disease. Treatment with IV oxytetracycline at a rate of 6.6 mg/kg of BW, q12h, is the treatment of choice. A response is usually seen within the first 12 h of therapy; including decreased rectal temperature, improved attitude and appetite, and a return of intestinal motility (12). Clinical signs frequently resolve in 3 to 5 d. However, oxytetracycline can cause antibiotic-induced colitis and should be used only in situations where PHF is highly suspected. Early diagnosis of PHF by the use of PCR further aids the practitioner’s decision on whether oxytetracycline therapy is warranted. Supportive care, such as IV fluids and nonsteroidal antiinflammatory drugs, should also be instituted should diarrhea develop. Signs of laminitis should be watched for and treated appropriately.

The natural route of infection for PHF is currently under investigation. Neorickettsia risticii was found to be closely realted to N. helminthoeca, the causative organism of salmon poisoning in dogs, which uses a helminth and aquatic snails as hosts. Neorickettsia risticii DNA was identified by using PCR in a helminth that infests aquatic snails and insects as its 1st and 2nd hosts, respectively (2). Horses experimentally inoculated with the helminth develop clinical signs of PHF, and N. risticii can be isolated from the infected horses’ blood and feces (11). An initial study indicates that ingesting aquatic insects that are secondary intermediate hosts of the helminth may be the natural route of infection (10). Damselflies, caddisflies, stoneflies, dragonflies, and mayflies have all been found to carry E. risticii-positive helminths (10). Transmission may occur if horses inadvertently eat dead flies in the grass while grazing, or dead insects can contaminate feed, water, or both; however, due to the small amount of horses in the study, other routes of transmission cannot be ruled out.

This report highlights the utility of using a PCR test on intestinal tissue to achieve a postmortem diagnosis of PHF. Veterinarians in the Maritimes should be aware that PHF exists in the region, so that they may consider it as a differential diagnosis. A survey of aquatic insects and snails in the area would help elucidate the epidemiology of the disease in the Maritimes.