On August 11, 2008, a 7-year-old racing Quarter Horse gelding was examined at a private veterinary clinic in Ocala, Fla, because the horse was lethargic, not eating or drinking, and had mild swelling in the limbs. The trainer indicated the horse was passing dark urine and had had a fever 3 days before the examination. Treatment given to the horse by the trainer prior to hospitalization included penicillin G procaine (6,600 U/kg [3,000 U/lb], IM, q 12 h), phenylbutazone (4.4 mg/kg [2.0 mg/lb], IV, q 12 to 24 h), and furosemide (0.88 mg/kg [0.40 mg/lb], IV, q 24 h). The horse's clinical signs had not improved after a day and a half of this treatment.

Further evaluation of the history revealed that the horse had been treated 6 months earlier for lameness; no further information on that incident was available. No vaccination history was available from the trainer except that the horse had not received any vaccines in the previous 12 months. The trainer also indicated that 3 other horses in his care on the index premises had swollen limbs and 1 additional horse appeared to be ill at the time of the visit, but details were lacking.

During initial examination at the veterinary hospital, the horse had a slightly high rectal temperature (38.39°C [101.1°F]), tachycardia (52 beats/min), and tachypnea (56 breaths/min). The mucous membranes were pale pink with a yellow tinge, and the sclerae were icteric. The horse's mentation appeared dull. Mild swelling was detected in the lower limbs and ventral abdomen, folliculitis was present over the entire body, and the horse voided dark brown, opaque urine.

The attending veterinarian initiated polyionic, isotonic fluid treatment IV and collected samples for a CBC, serum biochemical analysis, and urinalysis. Additional treatment included administration of trimethoprim-sulfamethoxazole (30 mg/kg [13.6 mg/lb], PO, q 12 h) and dexamethasone sodium phosphate (0.04 mg/kg [0.02 mg/lb], IV, q 24 h). The horse remained lethargic and mildly febrile and was observed to pass brown-colored urine but no manure during the first 24 hours of hospitalization.

The CBC and serum biochemical analysis findings included anemia (PCV = 23% [RI, 34% to 50%]), neutrophilia (8,362 neutrophils/μL [RI, 1650 to 7,475 neutrophils/μL]) with a slight left shift (226 band cells/μL [RI, 0 to 120 band cells/μL]), thrombocytopenia (52,000 platelets/μL [RI, 100,000 to 350,000 platelets/μL]), hyperfibrinogenemia (600 mg/dL [RI, 200 to 400 mg/dL]), azotemia (creatinine concentration, 11.8 mg/dL [RI, 1.2 to 2.9 mg/dL]; BUN concentration, 109 mg/dL [RI, 10 to 24 mg/dL]), hyperbilirubinemia (19.3 mg/dL [RI, 0 to 2 mg/dL]), hypocalcemia (7.7 mg/dL [RI, 11.2 to 13.6 mg/dL]), and hyperkalemia (5.1 mmol/L [RI, 2.4 to 4.7 mmol/L]). Urinalysis was performed by use of a dipstick test^a and gross and microscopic examination of a sample. Results included hemoglobinuria (3+) and bilirubinuria (3+), both with a range of possible values from 0 to 3+; proteinuria (2+), with a range of possible values from 0 to 3+; and aciduria (pH, 6.5). Diplococci and rod-shaped bacteria were evident on microscopic examination.

Initial differential diagnoses included EIA, EP, idiopathic immune-mediated hemolytic anemia, immune-mediated thrombocytopenia, purpura hemorrhagica, plant or chemical intoxication, and an allergic reaction to the previously administered penicillin. A serum sample was obtained by collecting blood in a red-top tube and allowing it to clot prior to centrifugation at 6,500 rpm, and samples were submitted for determination of Streptococcus equi M protein titers via ELISA^b (antibody titers were low [1:200]) and an ELISA test for EIA (which yielded negative results). Direct examination of a blood smear treated with a commercial Romanowsky-type stain^c was performed by the attending veterinarian at the clinic, and intracellular inclusions consistent with hemoparasites were detected within RBCs.

The attending veterinarian immediately sent the blood smear to the University of Florida College of Veterinary Medicine for further examination. The observed intracellular organisms were small to occasionally large, were oval to pear shaped, and had colorless to pale blue cytoplasm. These organisms were observed as single to multiple inclusions in multiple erythrocytes. The University of Florida clinical pathologist requested submission of a whole blood sample from the horse, and a repeated smear examined the next day confirmed the presence of similar inclusions. The morphology of the generally small, pleomorphic organisms was not consistent with Babesia caballi, but was consistent with Babesia equi. However, a previously unidentified Babesia sp or Theileria sp was also considered possible. The Florida State Veterinarian's office was contacted, and further testing was directed. A blood sample was collected and submitted to the NVSL for testing by means of cELISA^d as well as CFT and IFAT¹ The CFT and IFAT results were positive for antibodies against B equi, and the owner was given options for management of the horse via lifelong quarantine, euthanasia, donation to a research facility, or exportation out of the United States. The owner of the horse elected euthanasia.

A necropsy was performed by veterinary pathologists at the University of Florida College of Veterinary Medicine. Icterus and hemosiderosis were evident in multiple tissues. Tubular necrosis, likely caused by hemoglobinuria secondary to hemolytic anemia, was detected in the kidney. There was myeloid hyperplasia in the bone marrow. Hepatomegaly, likely due to congestion and accumulation of hemosiderin and hemosiderin-laden macrophages in the liver, was also observed. A DNA sample isolated from blood was amplified via PCR assays with piroplasm-specific primers² and the resulting amplicons were cloned and sequenced; presumptive identification of the hemoparasitic organism as B equi was made with a high level of confidence. Multiple frozen tissue samples and blood samples were shipped to the NVSL for evaluation by means of PCR analysis and serologic testing for antibodies against EP pathogens. Results of CFT, IFAT and PCR tests were positive for B equi.

The attending veterinarian reported the index case to the Florida State Veterinarian's office on August 13, 2008. The premises where the horse had resided prior to hospitalization (index premises [premises 1], Manatee County, Fla) was immediately quarantined by the State Veterinarian, and the USDA-APHIS office of veterinary services was notified. The index case was reported by the USDA-APHIS office of veterinary services to the OIE.

Activities conducted by Florida state veterinary officials included tracing of the movement of horses residing on the index premises, quarantine and testing of potentially exposed horses, tick surveillance, and an epidemiological investigation to determine the likely route of introduction and spread of the disease agent. Outreach to educate veterinarians, horse owners, and others in the equine industry regarding EP was also instituted and included a press release on August 15, 2008, concerning the detection of EP in the index case horse; advice was provided regarding clinical signs of the disease and avoidance of transmission, and individuals were requested to remain alert for signs of EP in horses in their care.

All testing of samples from horses as part of the regulatory response was conducted at the NVSL. Blood samples collected into red-top tubes (plain glass or serum separator) were centrifuged to obtain serum. Serum was tested for antibodies against B caballi and B equi by use of CFT, IFAT, and cELISA. During the outbreak, a horse was considered infected with an EP pathogen if it had a positive result for either the CFT or cELISA. The CFT and IFAT were performed according to the current version of the OIE-recommended protocols.¹ The cELISA^d for B caballi and B equi was performed according to the kit manufacturer's directions except that all serum samples were tested in duplicate.

Whole blood samples collected into tubes containing EDTA were also submitted to the NVSL for PCR assays. Genomic DNA was extracted from 100 μL of blood by use of a commercial kit^e according to the manufacturer's directions, with the addition of a 30-minute incubation step at 56°C with 50 μL of a 2 mg/mL solution of proteinase K added to the cell lysis solution. The nested PCR assay to test for B equi was performed as previously described³ with modifications to improve specificity by increasing the binding primer dynamics. Primers used were external forward 5′-GAGGAGGAGAAACCCAAG-3′, external reverse 5′-GCCATCGCCCTTGTAGAG-3′, internal forward 5′-TCAAGGACAACAAGCCATAC-3′, and internal reverse 5′-TTGCCTGGAGCCTTGAAG-3′. Cycling conditions for the external reaction were initial denaturation at 95°C for 5 minutes; 30 cycles of 95°C for 20 seconds, 60°C for 20 seconds, and 72°C for 20 seconds; and a final elongation of 72°C for 5 minutes with a 4°C hold. Internal cycling conditions were an initial denaturation at 95°C for 5 minutes; 30 cycles of 95°C for 5 seconds, 60°C for 5 seconds, and 72°C for 5 seconds; and a final elongation of 72°C for 5 minutes with a 4°C hold. Nested PCR for B caballi was performed as previously described.⁴ Twenty microliters of each internal reaction product was run on 2% agarose gels^f containing ethidium bromide and visualized by use of an imaging system.^g

Thin blood films were prepared from whole blood in EDTA, fixed in methanol, and stained with Giemsa. The slides were screened at 630× magnification for blood parasites, which were confirmed at 1,000× magnification. Identification of parasites was based on morphological features detectable by use of light microscopy.

In addition to quarantine of the premises where the index horse was housed, 3 properties with horses located adjacent to the index premises were quarantined. All horses on these 4 premises were tested for EP via serologic (cELISA, IFAT, and CFT), PCR, and whole blood smear evaluations. An investigation was started to determine if EP had spread beyond the immediate area of the index premises. At the time of the initial investigation, the method of introduction and means of transmission of B equi among horses in Florida were unknown. In an effort to determine the source of the outbreak, movement of all horses onto and off of the index premises was traced to the greatest extent possible.

Each individual property in which 1 or more horses tested positive for infection with an EP pathogen was considered an infected premises and was assigned a number on the basis of the order of information gathered during the investigation (Table 1). On each infected premises, interviews were conducted to determine the movement of all horses onto and off of the premises for as long as could be ascertained. Tracing information was based on recollections of the owner and staff and on available records for that premises. When a new premises was identified from which horses with EP had arrived or to which horses from an infected premises had been moved, the new location was quarantined by the state while an investigation was undertaken. The emphasis of the tracing effort was to identify premises where previous movement of any of the B equi—infected horses may have led to exposure of additional resident horse populations.

As part of the surveillance activities, all horses that were considered potentially exposed to EP pathogens on the basis of epidemiological investigations were retested 60 days after the last known possible exposure. On premises where horses with EP were identified, the exposure risk was considered to have been eliminated once all infected horses were removed from that premises. Criteria for quarantine release included the following: any horses with positive test results had to be removed from the premises a minimum of 60 days prior to retest of exposed horses; all horses remaining on the premises had to test negative for antibodies against B equi and B caballi by means of both the CFT and cELISA; no exotic ticks could be found on the premises; and no EP agents could be detected in domestic ticks found on the premises. Owners of horses that had positive test results were offered retesting to confirm infection and were given the options of having the infected horse quarantined for life, euthanized, shipped to a research facility in the United States, or exported to another country.

Some of the exposed premises housed horses used in nonsanctioned racing (ie, race competitions not approved by any recognized regulatory racing authority), and others did not. On premises where horses were involved in nonsanctioned racing, all horses present were tested for EP; on the remaining premises, only the horses that had been traced to the premises were initially tested. If 1 or more traced horses tested positive, all horses on that premises were then tested.

In total, 210 horses were tested for infection with EP pathogens during the outbreak investigation. No horses were found to be infected with B caballi, and 20 tested positive for B equi infection. At the time of the initial quarantine of the index premises, 4 horses had clinical signs that were similar to those of the index horse. These horses were being treated by the trainer and tested positive for B equi. Two additional B equi–infected horses were observed to have clinical signs consistent with chronic EP (pale mucous membranes, reduced appetite, and poor body condition) at the time of euthanasia. All horses that tested positive were Quarter Horses from 3 to 12 years of age, and these included 8 mares, 2 stallions, and 10 geldings. Horses were quarantined and tested on 25 premises in 9 different counties, and ≥ 1 B equi—infected horse was identified on 7 of these premises (premises 1 through 7; Table 1). The number of horses with positive test results ranged from 1 to 6/premises.

During the initial investigation on premises 1 and 2, all horses (n = 50) were tested by means of CFT, cELISA, IFAT, PCR, and examination of a direct blood smear. After reviewing test results for horses on premises 1 and 2, the PCR assay was not part of the testing performed at other locations unless a horse tested positive for B equi infection via ≥ 1 other method. When the initial 50 horses were first tested via all 5 methods, 3 horses had weakly positive PCR test results, observed as faint bands in agarose gels. These results were not supported by the serologic or direct smear diagnostics as the results of the cELISA, CFT, IFA, and direct-smear examinations were negative for B equi. These 3 horses were retested 25 days later via the described serologic tests with negative results and were considered to test negative for B equi infection for purposes of the outbreak investigation. The findings for these 3 horses, along with the need to expedite testing on other potentially infected horses, led to the decision to eliminate PCR testing on the remaining horses unless they tested positive via ≥ 1 other method. Direct smears of samples from horses with negative serologic test results on premises 6 and several other horses with weak epidemiological links to B equi–infected horses were not evaluated because infection in these horses was considered unlikely on the basis of results from other tests and findings of the epidemiological investigation.

Of 20 horses determined to be infected with B equi, 13 tested positive via both cELISA and CFT, 4 tested positive via cELISA but negative via CFT, and 3 tested positive via CFT and negative via cELISA. Examination of direct blood smears from 5 horses yielded positive results. Neither cELISA nor CFT alone detected B equi in all infected horses. Three horses that tested positive via CFT but negative via cELISA had the causative organisms identified on a direct blood smear, indicative of a relatively high level of parasitemia and possibly a recent exposure to B equi.

Owners had the option to request retesting of horses that had positive results for B equi infection on initial testing. If a retest was requested by the owner, all 5 of the previously described tests were performed. Six of the 20 horses that initially tested positive were retested at the owner's request, and all 6 had positive results for ≥ 1 serologic test.

Only 1 horse that initially tested negative for B equi infection subsequently tested positive on the 60-day postexposure test. This horse was located on premises 2 and had positive test results via cELISA, IFA, CFT, and examination of a whole blood smear at the time of postexposure evaluation; when the horse was tested again 1 week later, results for all 5 tests (including PCR assay) were positive. This horse was considered to have been exposed to EP on its current premises because it had been at that location for several years and initially had negative results for all tests. In addition, the test results were consistent with recent infection. After detection of infection with the 60-day postexposure test on premises 2, state animal health officials implemented postexposure testing at 30 days in addition to the 60-day postexposure-testing protocol on the 3 remaining quarantined premises (2, 3, and 6) in an effort to detect any acute infections in a more timely manner.

During the investigation, 420 contacts were made by State of Florida personnel, requiring > 4,000 hours of work. The total cost borne by the state to complete the investigation was estimated at $158,000.

There were no domestic restrictions in the United States on movement of horses from Florida except for horses residing on the quarantined premises. However, restrictions were imposed on the exportation of horses to Canada. The week of September 12, 2008, Canadian officials notified the USDA that Canada would no longer accept horses that had been in Florida during the 21 days prior to exportation. On October 10, 2008, on the basis of apparent containment of the EP outbreak, Canadian importation requirements were revised. Specifically, exportation of horses from Florida to Canada required that the Canadian importer have an import permit, and each horse had to be inspected by a veterinarian ≤ 15 days prior to the date of importation. A statement on the export health certification regarding lack of EP exposure was also required, and horses had to have tested negative for antibodies against EP pathogens via cELISA (or, where applicable, to have tested negative via an alternate test acceptable to Canadian officials) during the 15 days prior to date of importation. In addition, health certifications issued after September 15, 2008, for Canadian importation of horses from other states in the United States had to indicate that the horses had not been in Florida during the 21 days prior to exportation.

During the epidemiological investigation, personnel at the Florida State Veterinarian's office received verbal reports from those interviewed, including owners, trainers, grooms, and other farm personnel, that 3 horses from premises 3 had been imported from Mexico. One of these horses was confirmed to have been imported from Mexico in May 2005 through the USDA Laredo import center and tested negative for B equi infection by means of CFT. Two additional horses had reportedly come from Mexico in 2004 or 2005, but the specifics about this movement were unavailable and could not be confirmed on the basis of official import records. Both of these horses tested positive for B equi and appeared to have been involved in the spread of B equi on the basis of their traced movements to other premises. These 2 horses were presumed to be the source of B equi in the outbreak; they had moved from premises 3 to premises 4 and 5, from which exposed horses had subsequently relocated to 1 or more of the other 4 infected premises (Figure 1).

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Presumed timeline and sequence of transmission of Babesia equi infection that resulted in an outbreak of EP in Florida in 2008. Arrows indicate the known movement of B equi–infected horses among premises. Three horses were imported from Mexico to premises 3 early in 2005. Two of these 3 horses tested positive for B equi infection and were believed to be the source of infection at premises 3, 4, and 5, with infection spread to the remaining premises by subsequent movement of other infected horses. *One of 4 horses at premises 5 that became infected was later serially relocated to premises 6, 2, and 1 and was presumed to be the source of infection at all 3 premises. PT = Presumed period of transmission.

Citation: Journal of the American Veterinary Medical Association 240, 5; 10.2460/javma.240.5.588

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Investigation by personnel from the Florida State Veterinarian's office revealed that the horses infected with B equi were all involved in nonsanctioned racing. Persons involved in the care of these horses implied that practices such as use of shared needles and syringes, administration of illicit drugs, and a procedure termed blood packing or blood doping (collection of blood from one horse that is then injected into another horse prior to competition) had taken place. Since blood transfer was not performed by licensed veterinarians, it was considered likely that the donor horses were not tested for the presence of blood-borne disease agents or for compatibility with the recipient based on crossmatching.

Horses present on all 25 premises involved in the epidemiological investigation were examined for ticks at the time of the initial investigation. In addition, surveys for ticks on wild mammals were conducted at selected premises by the Southeastern Cooperative Wildlife Disease Study, University of Georgia. Intense tick surveillance was conducted on 10 premises. This included the 7 premises where infected horses were identified and 3 premises with horses that were adjacent to premises 1. On premises with B equi–infected horses, tick surveillance included examination of horses; use of CO₂ traps; tick drags, which involved dragging white sheets through vegetation to collect the ticks; examination of other domestic animals on the premises; and live-trapping, examination, and subsequent release of wild mammals. On adjacent premises, surveillance included repeated examinations of horses during site visits and trapping and examination of wild mammals.

Tick collection was performed from August 13 through December 8, 2008. The tick surveillance period at each location varied according to availability of personnel and equipment, presence of vegetation, availability of wildlife habitat, and abundance of wildlife. Surveillance for ticks on wild mammals was conducted for 1 week on each of 2 premises, for 2 weeks on 7 premises, and for 4 weeks on 1 premises. The surveillance period was reduced to 1 week on 2 premises because vegetation, wildlife habitat, and wildlife populations were considered minimal. Surveillance was extended to 4 weeks on 1 premises because of adequate wildlife habitat and wild host abundance and because specimens of Dermacentor variabilis were found during the second week of trapping.

Three species of ticks were found on premises where surveillance was conducted: D variabilis, Amblyomma maculatum, and Ixodes scapularis. The number and species of ticks collected per premises varied; no ticks were found on some premises and 522 were collected on 1 premises. This variation was attributed to differences in the density of ticks on the selected premises. No ticks were found on B equi–infected horses during the investigation. After collection, all recovered ticks were shipped to the NVSL for identification and specimens of D variabilis (the only tick species collected that was considered a potentially competent vector for B equi) were tested for the presence of EP pathogens by means of PCR assay. All 64 specimens of D variabilis recovered tested negative for B equi.

In summer 2009, follow-up tick surveillance was conducted by the Southeastern Cooperative Wildlife Disease Study group on 3 premises that had housed infected horses in 2008. Surveillance involved wild mammal trapping with tick examination of the animals prior to release. No exotic tick species were recovered, and all specimens of D variabilis collected tested negative for B equi.

All B equi—infected horses were euthanized (n = 17), died (1), or were shipped to a US federal facility for research purposes (2). The horse that died was reported to have been found dead in its stall. The cadaver was delivered to the Kissimmee Animal Disease Diagnostic Laboratory for necropsy. No cause of death was determined. At the end of December 2008, only 2 premises remained under quarantine to allow for retest of horses at 60 days after exposure. In February 2009, the quarantine was lifted on these remaining premises.