Granulocytic anaplasmosis in a horse from Saskatchewan

Abstract

This report describes a case of equine granulocytic anaplasmosis in a horse from Saskatchewan. Morulae were visualized within blood neutrophils, and the diagnosis was confirmed by polymerase chain reaction (PCR). The organism was identified as the human pathogenic strain of Anaplasma phagocytophilum by PCR and DNA sequencing of 3 independent genes.

A 30-year-old Arabian-cross gelding was presented at the end of October 2010 with a primary complaint of inappetence. On physical examination, the horse was found to be in moderate body condition (BCS 2.5/5) with normal rectal temperature, heart rate, and respiratory rate. Mucous membranes were markedly icteric and the left submandibular lymph node was enlarged. There were no other significant physical abnormalities. Reportedly, the horse had been purchased from Alberta 19 y previously and had never been transported outside of Canada. The horse had lived on the same farm in Saskatchewan since it was purchased and had only occasionally traveled within the province. The only other horse on the farm was also examined, but it did not show any abnormalities. Ethylenediamine tetra-acetic acid (EDTA) blood and serum were submitted to Prairie Diagnostic Services (Saskatoon, Saskatchewan) for a complete blood (cell) count (CBC), serum biochemistry, and testing for equine infectious anemia. The erythron parameters were within reference intervals when analyzed by the Cell Dyn 3500R hematology analyzer (Abbott Diagnostics, Abbott Park, Illinois, USA), and total leukocyte numbers were moderately decreased [3.4 × 10⁹/L; reference interval (RI): 4.3 to 11.5 × 10⁹/L]. Leukopenia is a common hematologic finding in cases of Anaplasma phagocytophilum infection, typically reflected as both a neutropenia and lymphopenia.

As the EDTA blood sample was aged due to transport, an accurate differential white cell count was not possible and platelet numbers were considered unreliable. Examination of a blood smear revealed the presence of a low number of poorly preserved leukocytes that contained basophilic coccoid organisms arranged both haphazardly within the cytoplasm, and occasionally as morulae. Serum biochemistry (Hitachi 912; Roche Diagnostics, Basel, Switzerland) revealed a mild hyponatremia (129 mmol/L; RI: 132 to 142 mmol/L), moderate hypophosphatemia (0.36 mmol/L; RI: 0.53 to 1.19 mmol/L), mild hypomagnesemia (0.54 mmol/L; RI: 0.66 to 1.20 mmol/L), and a mild hyperbilirubinemia (73 μmol/L; RI: 2.0 to 41 μmol/L) due to an elevated indirect fraction. Most of the serum biochemistry abnormalities could be attributed to the history of anorexia. The test for equine infectious anemia was negative. As the original EDTA sample was poorly preserved, a fresh blood smear was submitted 3 d after the original submission to examine for the presence of microorganisms. On this smear, leukocyte numbers (6 × 10⁹/L; RI: 4.3 to 11.5 × 10⁹/L) and the differential count were estimated to be within reference intervals, as were platelet numbers (115 × 10⁹/L; RI: 100 to 600 × 10⁹/L). Single, rarely 2, granular basophilic morulae consistent with A. phagocytophilum, were noted in approximately 15% of the neutrophils. The EDTA blood was submitted for polymerase chain reaction (PCR) (Vector Borne Disease Diagnostic Lab, North Carolina State University, Raleigh, North Carolina, USA) to confirm the presence of A. phagocytophilum. Given the potential for co-infection with Borrelia burgdorferi, serum was also submitted for Lyme indirect fluorescent antibody (IFA) serology (Idexx Laboratories, North Grafton, Massachusetts, USA). The PCR analysis was positive for A. phagocytophilum, and the IFA test for Lyme disease showed a titer of 1:200 (> 1:100 is considered positive).

Subsequently, molecular analyses were conducted (Biology Department, University of Saskatchewan) to determine which strain of A. phagocytophilum was present in the horse blood. Genomic (g) DNA was prepared from the horse blood using the DNeasy Blood & Tissue Kit (Qiagen, Toronto, Ontario) and subjected to PCR analyses using 3 genetic markers; the 16S rRNA gene (1), the ankyrin protein A (ankA) gene (2) and the citrate synthase (gltA) gene (3). Amplicons of each gene were subjected to automated DNA sequencing and the sequences obtained were compared to sequence data from GenBank. Our sequences of the 16S, ankA and gltA genes were 100% identical to the sequences of the corresponding genes for a human pathogenic strain of A. phagocytophilum (GenBank accession nos. U02521, AF10893, and AF304136, respectively). The genomic DNA (gDNA) derived from the horse blood was also tested for the presence of B. burgdorferi using a nested PCR analysis targeting the ospA gene(4); however, no PCR-positive products were obtained.

At the time of laboratory detection of A. phagocytophilum in the blood, the patient showed clinical improvement; therefore, no therapy was initiated. Follow-up was obtained 7 mo after diagnosis, at which time the horse had improved its body condition (BCS 3/5) and showed good appetite. Mucous membranes still appeared moderately icteric. Routine blood work was repeated and showed the following abnormalities: serum biochemistry (Hitachi 912) revealed a mild hyperglobulinemia (41 g/L; RI: 21 to 39 g/L), suggesting inflammation or chronic antigenic stimulation, and mild hyperphosphatemia (1.24 mmol/L; RI: 0.53 to 1.19 mmol/L) with a mild monocytosis (0.63 × 10⁹/L; RI: 0.1 to 0.5 × 10⁹/L) on CBC (Cell Dyn 3500R), suggesting increased tissue demand for macrophages. No morulae were found on evaluation of the blood smear. Another PCR analysis (Vector Borne Disease Diagnostic Lab) conducted on blood taken at this time was negative for A. phagocytophilum. Similarly, IFA serology for A. phagocytophilum (Diagnostic Centre for Population and Animal Health, Michigan State University, Lansing, Michigan, USA) showed a titer of < 80 which is considered negative (> 160 is considered positive). As there was no evidence for ongoing A. phagocytophilum infection, the suggestion of inflammation on repeat bloodwork was presumed to be unrelated to the initial presenting complaint. An IFA titer obtained from the other horse on the premises (which had not shown any clinical signs compatible with anaplasmosis) was also negative (< 80).

Anaplasma phagocytophilum is an obligate intracellular coccoid to ellipsoid bacterium that can infect humans and domestic animals, including dogs and horses. This tick-borne pathogen is the causative agent of equine granulocytic anaplasmosis (EGA). In the United States, the principal vectors of A. phagocytophilum are Ixodes scapularis in the midwest and northeast and I. pacificus in California (5). Although EGA has been well-described in horses in North America (5–9), we are reporting the first case of the disease in Saskatchewan, and to our knowledge, only the third case in Canada (6,7). Previous case reports of EGA in Canada did not identify the strain(s) of the causative agents. However, in the present case, PCR and DNA sequencing of 3 independent genes identified the presence of a human pathogenic strain (Ap-ha) of A. phagocytophilum in the gDNA extracted from the blood of the horse. The Ap-ha strain has been reported in human patients and in I. scapularis from the upper midwest and northeast of the United States (1,10,11), and in the blood of infected horses from Minnesota and Connecticut (8,9). Horses in California are infected by different strains of A. phagocytophilum (5).

Although there are no known established populations of either vector in Saskatchewan, adult I. scapularis have been collected in the province by passive surveillance (12). Migrating passerine birds from the United States are known to transport I. scapularis larvae and nymphs into Canada (4). Geographically isolated populations of I. scapularis have recently become established in several localities in southern Canada, including on the north shore of Lake Erie and Lake Ontario, in Nova Scotia, and in south-eastern Manitoba (12). It has been predicted that environmental conditions may become more suitable in the future for the establishment of I. scapularis populations in Saskatchewan as a consequence of climate change (13), which may increase the risk of exposure to pathogens carried by this tick species. Granulocytic anaplasmosis has been identified in 3 dogs from Saskatoon, Saskatchewan which, like the gelding in this case, had no history of travel outside of the province (14). This suggests that each of these infections was acquired from infected I. scapularis in the province. Ticks had reportedly never been noticed on the gelding referred to in this report. The gelding also had a positive titer for B. burgdorferi, and while co-infection of I. scapularis with B. burgdorferi and A. phagocytophilum has been reported, it is rare in Canada (1). Therefore, the gelding was most likely exposed to 2 or more I. scapularis prior to diagnosis. Presence of B. burgdorferi in blood could not be detected by PCR in this case; however, this was not unexpected as this bacterium is rarely detected in this type of sample. A convalescent titer may have assisted in discriminating previous from recent exposure to B. burgdorferi, but was not pursued.

The gelding in this report had clinical and laboratory signs associated with EGA including anorexia, icterus, leukopenia, hyperbilirubinemia, and circulating morulae. Although the disease can be self-limiting in 2 to 3 wk, therapy with oxy-tetracycline has typically been instituted in reported cases of EGA (6,7). In the case reported here, clinical improvement was noted at the time of laboratory identification of EGA; therefore, therapy was not instituted. The gelding continued to improve and, with the exception of mild icterus, clinical signs resolved. Presence of icterus upon re-evaluation was not easily explained in this case but, as hyperbilirubinemia was no longer present, may have been attributable to dietary factors such as high carotene intake on spring pasture. Seven months after initial presentation, PCR analysis and serology for A. phagocytophilum were negative, suggesting clearance of the infection. Antibody titers have been reported to persist for as short a time as 3 mo post-infection (15).

The case reported here reinforces that, although rare, EGA should be considered as a differential diagnosis for horses with compatible clinical signs living in Saskatchewan or other non-endemic regions of Canada. In addition, although treatment with tetracycline has frequently been reported in confirmed cases of EGA, this case seems to support the potentially self-limiting nature of the disease. CVJ