Clinical Presentation of 26 Anaplasma phagocytophilum-Seropositive Dogs Residing in an Endemic Area

Introduction

Anaplasma (A.) phagocytophilum is the etiological agent of canine granulocytic anaplasmosis (CGA), formerly known as granulocytic ehrlichiosis. In 2001, the order of Rickettsiales was reorganized after genomic analysis of 16S ribosomal ribonucleic acid, groESL, and surface protein genes indicated that previous classifications were incorrect. Ehrlichia (E.) equi, E. phagocytophila, and the agent causing human granulocytic ehrlichiosis were ultimately determined to be one organism now known as A. phagocytophilum.¹ Anaplasma phagocytophilum has received considerable attention over the last 2 decades, as it is capable of inciting moderate to severe clinical disease in a variety of mammals—most notably humans, dogs, and horses. The bacterium was first documented in humans in Minnesota and Wisconsin in 1990, and soon after it was recognized in dogs.^2,3

Anaplasma phagocytophilum is an obligate intracellular, gram-negative, aerobic bacterium that infects neutrophils and, more rarely, eosinophils. It is transmitted by Ixodes scapularis ticks in the midwest and the northeastern United States and by Ixodes pacificus ticks in the northwestern United States. Anaplasma phagocytophilum is maintained in the wild by reservoir hosts, including white-tailed deer and small rodents such as the white-footed mouse.^4,5 The disease is endemic in the upper midwest, the northeastern United States, and the western United States from California to British Columbia; but it has also been reported throughout the United States, Europe, and a few countries in Asia.

Previous studies have documented a seasonality to clinical cases that correlates with the feeding behavior of the Ixodes vectors. In the upper midwest, for example, most dogs with CGA have been reported to be presented either in early summer (May and June) or in the fall (October and November).^3,6 Middle-aged or older female dogs may be at higher risk of infection, although this finding has not been consistent.³

The presenting clinical signs of CGA are varied and often nonspecific. The clinical picture is often further complicated by coinfection with Borrelia (B.) burgdorferi sensu stricto, the causative agent of Lyme disease in North America, which is also transmitted by Ixodes ticks.^7,8 One study showed that infection with both A. phagocytophilum and B. burgdorferi simultaneously is more likely to result in clinical disease and more severe clinical signs; however, determination of whether specific clinical signs are associated with coinfection has not been made.⁶ In a large survey of dogs that were seropositive to A. phagocytophilum, B. burgdorferi, and Ehrlichia species, the most commonly reported clinical signs included fever, lethargy, and musculoskeletal pain or discomfort.⁶ Experimentally induced infections with A. phagocytophilum have yielded similar results, with fever, depression, and inappetence being the most common signs of infection.⁹ Other, less commonly reported clinical signs of CGA include gastrointestinal upset (i.e., vomiting and/or diarrhea), hepatomegaly, splenomegaly, lymphadenopathy, polydipsia, central nervous system abnormalities, and epistaxis.^3,6,10,11

The most consistent laboratory abnormalities reported in dogs with CGA are thrombocytopenia and lymphopenia.^3,6,12,13 Nonregenerative anemia has also been seen in a number of clinically ill dogs.^9,14,15 One case of immune-mediated hemolytic anemia (IMHA) was reported in a mixed-breed dog diagnosed with CGA in the United Kingdom.¹⁰ Serum biochemical abnormalities frequently noted in dogs with CGA include moderate hypoalbuminemia and increased serum alkaline phosphatase (ALP) activity.^3,14,15

Dogs seropositive for antibodies to A. phagocytophilum are common in endemic areas;⁶ however, attributing clinical signs to active infection can be difficult, because morulae only circulate for 4 to 8 days within the first 2 weeks after transmission.⁹ Thus, confirming active infection with routine diagnostic screening is not always possible. Controversy is present over the existence of chronic granulocytic anaplasmosis infection in dogs. Although chronic monocytic ehrlichiosis due to E. canis infection has been documented in dogs, chronic granulocytic anaplasmosis has only been shown in humans and sheep and has never been convincingly demonstrated in dogs.^16–19

The primary aims of this study, therefore, were to describe the range of clinical signs in A. phagocytophilum-seropositive dogs that were presented to the Veterinary Medical Teaching Hospital (VMTH) at the University of Wisconsin-Madison and to examine the prevalence of IMHA in this population of dogs. Furthermore, we wanted to evaluate whether specific clinical signs were associated with coexposure to B. burgdorferi in actively infected dogs. A final aim of this study was to determine whether the clinical response to doxycycline was complete in all treated dogs.

Materials and Methods

Dogs that were seropositive for antibodies to A. phagocytophilum were identified by a review of the clinical pathology database (Universal Veterinary Information System) at the University of Wisconsin-Madison VMTH over a 3.5-year period, from August 2004 through March 2008. All serological testing was performed by the same commercial laboratory^a using an indirect fluorescent antibody (IFA) method directed against fixed equine neutrophils containing the organism.

Criteria used for inclusion in the study were seropositivity for A. phagocytophilum (titer≥1:80), clinical illness, complete medical record information, and adequate information on treatment response over the 4 weeks after doxycycline initiation. Medical record information included signalment; body weight; town and county of residence; complete history (including history of tick exposure and travel history); physical examination findings; results of complete blood count (CBC), serum biochemical profile, and urinalysis; results of serological testing for B. burgdorferi, E. canis, Rickettsia (R.) rickettsii, E. risticii, and Babesia canis (when performed); and prescribed treatment (e.g., drugs, dosage, duration, and response).

Ehrlichia canis, R. rickettsii, E. risticii, and Babesia canis testing was performed using IFA tests on sera from the dogs.^a Borrelia burgdorferi antibodies were detected using one or more different testing modalities in each dog, including serum enzyme-linked immunosorbent assay (in four of 11 dogs),^b IFA testing^a (in six of 11 dogs), and Western blot^a (in one of 11 dogs). Additional tests, such as thoracic radiography, abdominal ultrasonography, and urine or blood cultures were documented when performed.

Peripheral blood smears from all available dogs were reviewed retrospectively by a single clinical pathologist (Young) for the presence of granulocytic morulae. Leukocytes were systematically examined on each smear, with particular attention paid to leukocytes at the feathered and side edges of the smears. Response to the prescribed therapy was recorded and was based on review of the medical record as well as telephone contact with the dog’s owner or primary veterinarian during the summer and fall of 2008.

Data between groups of dogs, such as the prevalence of specific clinical signs between A. phagocytophilum-seropositive dogs with and without seropositivity for antibodies to B. burgdorferi, were compared using a Mann-Whitney U test, with P<0.05 considered significant.

Results

The medical records of 35 dogs that were presented to the University of Wisconsin-Madison VMTH and were seropositive for antibodies to A. phagocytophilum were reviewed. Twenty-six dogs seropositive to A. phagocytophilum met inclusion criteria. Nine dogs were excluded from the study for the following reasons: the antibody titer to A. phagocytophilum was <1:80, case information was insufficient, no clinical signs were present, or a convincing and definitive alternate diagnosis accounted for the clinical signs.

Twelve (46%) of the 26 included dogs had positive antibody titers for A. phagocytophilum only, and 45% (10 of 22 tested for both organisms) had positive titers for both A. phagocytophilum and B. burgdorferi only. Of the 10 dogs that had positive B. burgdorferi titers, three were vaccinated for B. burgdorferi prior to documentation of positive titers, and seven were not vaccinated. The three dogs that were vaccinated received their vaccines within 10 months of their positive Lyme titer. The vaccination status of the dogs that were not seropositive for B. burgdorferi was not documented. Positive titers were reported for other organisms, including E. canis (8.3%; two of 24 tested), R. rickettsii (4.8%; one of 21 tested), and E. risticii (9.0%; one of 11 tested).

Antibody titers to A. phagocytophilum ranged from 1:80 to 1:40,960 (median titer 1:1280). Of the 26 dogs seropositive for A. phagocytophilum, 12 were female (11 spayed, one intact) and 14 were male (12 neutered, two intact); no sex predisposition was found. Most of the dogs were large-breed varieties. Body weight ranged from 11 to 62.2 kg (median 30.1 kg). Six of the dogs were mixed breeds, and 20 were purebred. Labrador retrievers were the most common breed seropositive for A. phagocytophilum (15%; four of 26); however, this rate was comparable to the percentage (12.5%) of Labrador retrievers presenting to the VMTH overall during this same time period (suggesting that no breed predilection was present). No other breed had more than two dogs represented. The median age was 5.2 years (range 1.7 to 14.5 years). Granulocytic morulae were not detected on peripheral blood smears in any of the 26 dogs included in this study.

The counties of residence for the 26 dogs are shown in Figure 1. The Wisconsin counties represented included Dane, Columbia, Outagame, Portage, Jefferson, Waukesha, Rock, Iowa, Crawford, La Crosse, Wood, Lincoln, Barron, Washburn, Winnebago, and Vilas. Fifteen of the 26 dogs lived in the southern third of Wisconsin, and eight of those dogs were from Dane County where the University of Wisconsin-Madison VMTH is located. Eight of the 10 dogs seropositive to A. phagocytophilum alone had no history of recent travel outside of the state; the owners of the two other dogs in this group could not be reached for a review of travel history.

The prevalence of clinical signs and physical examination findings in all 26 A. phagocytophilum-seropositive dogs are summarized in Figure 2. Lethargy (81% of dogs), inappetence (58%), and lameness (50%) were the most common clinical signs, followed by fever (46%). Two dogs were presented with cough, and two dogs had epistaxis. Other significant clinical signs or physical examination findings included lymphadenopathy, joint effusion, splenomegaly, hepatomegaly, and vomiting.

Prevalence rates of laboratory abnormalities for the 26 A. phagocytophilum-seropositive dogs are listed in the Table. Thrombocytopenia was the most common laboratory abnormality and was found in 56% of dogs. Leukocytosis from a mature neutrophilia and a regenerative left shift was observed in 28% of dogs. Approximately 25% of dogs had increased serum ALP activity. Hypoalbuminemia and proteinuria were seen in 27% and 17% of dogs, respectively.

Notably, IMHA was diagnosed in three dogs based on the presence of regenerative anemia with 3+ spherocytosis and/or a positive direct Coombs’ test. Two of these dogs were seropositive for antibodies to other agents: E. risticii in one case (negative for Babesia spp., B. burgdorferi, E. canis, and R. rickettsii) and B. burgdorferi and R. rickettsii in the other case (negative for E. canis). All three dogs had abdominal imaging (ultrasonography or radiography), and two of the three dogs had thoracic radiography. None of the imaging revealed evidence of neoplasia as a trigger for the IMHA. All three dogs with IMHA died or were euthanized for progressive disease.

One dog with IMHA did not have serological evidence of infection with tick-borne agents other than A. phagocytophilum. This dog was an 8-year-old, spayed female, Labrador mix with a prior history of fever, episcleral injection, inappetence, lethargy, and panting noted 2 months prior to presentation. A CBC, serum biochemical panel, urinalysis, and abdominal radiography at that time revealed mild thrombocytopenia (168,000/μL; range 175,000 to 500,000/μL) with subjectively enlarged platelets, a normal hematocrit (HCT), clinically insignificant proteinuria (100 mg/dL with a urine specific gravity of 1.060), and splenomegaly. Serological testing for antibodies to B. burgdorferi and for heartworm antigen was negative. Testing for A. phagocytophilum and other tick-borne rickettsial organisms was not done at that time; however, the dog responded promptly to doxycycline (6 mg/kg per os [PO] q 12 hours) and amoxicillin (15 mg/kg PO q 12 hours), both administered for 5 weeks.

Approximately 2 weeks after discontinuation of antibiotics, lethargy and inappetence recurred, along with exercise intolerance and episodes of collapse. Physical examination by the primary veterinarian revealed pallor, fever (103.2°F), and tachycardia. Laboratory testing revealed a severe regenerative anemia (HCT 19%; range 37% to 55%) with hyperbilirubinemia (1.9 mg/dL; range 0.1 to 0.5 mg/dL). Thoracic radiographs were unremarkable, and abdominal radiographs revealed moderate hepatosplenomegaly. The direct Coombs’ test was positive, and treatment with an immunosuppressive dosage of prednisone (1 mg/kg PO q 12 hours) was initiated. Azithromycin (15 mg/kg PO q 24 hours) was also instituted. The dog became icteric, dyspneic, and recumbent, and it was referred to the University of Wisconsin-Madison VMTH. Serological testing for E. canis, E. risticii, Babesia gibsoni, Babesia canis, and B. burgdorferi was negative. The serum antibody titer to A. phagocytophilum was 1:640. Abdominal ultrasonography showed no evidence of neoplasia. Blood and urine cultures and an echocardiogram showed no evidence of underlying bacteremia or endocarditis. Despite aggressive supportive therapy (including packed red blood cell transfusions, plasma, and heparin), the dog developed disseminated intravascular coagulation (DIC) and presumptive pulmonary thromboembolism and was euthanized.

All dogs seropositive only for A. phagocytophilum (n=12) were compared with dogs seropositive for antibodies to only A. phagocytophilum and B. burgdorferi (n=10). None of the 12 dogs seropositive to A. phagocytophilum alone had platelet counts <105,000/μL (median platelet count for the six thrombocytopenic dogs in this group was 144,000/μL; range 105,000 to 173,000/μL). Dogs that were thrombocytopenic (five of 10 dogs in this group) and with antibodies to both A. phagocytophilum and B. burgdorferi had a median platelet count of 51,000/μL (range 20,000 to 171,000/μL), which was significantly lower than the platelet counts in dogs with antibodies only to A. phagocytophilum (P=0.04). Data were insufficient to compare the prevalence rates of proteinuria between dogs seropositive only to A. phagocytophilum and dogs seropositive for antibodies to both A. phagocytophilum and B. burgdorferi. Seven of these 22 dogs did not have a full urinalysis or urine protein:creatinine (UPC) ratio performed. The only dogs with a UPC >1.0 were positive for antibodies to both A. phagocytophilum and B. burgdorferi.

Nine of 12 dogs seropositive to A. phagocytophilum alone responded to a single course of doxycycline. Dosages ranged from 4.9 to 7.7 mg/kg q 12 hours to 11 mg/kg q 24 hours, and duration of treatment ranged from 21 to 28 days. Of the three dogs without a prompt clinical response, one dog (highlighted previously) developed IMHA 2 weeks after stopping a 5-week course of doxycycline for suspected CGA. A second dog with lethargy, inappetence, pelvic limb stiffness, and cough failed to respond completely to a 3-week course of doxycycline (5.8 mg/kg PO q 12 hours). Stiffness and lethargy resolved after a second course of doxycycline was initiated (8.7 mg/kg PO q 12 hours for 28 days). Other causes for these clinical signs were considered less likely, as serological and polymerase chain reaction (PCR) results were negative for other tick-borne organisms (R. rickettsii, E. canis, Babesia gibsoni, Bartonella), and the CBC, serum biochemical panel, and abdominal ultrasonography findings were all normal. Canine pancreatic lipase immunoreactivity was mildly increased (236 μg/L; reference interval <200 μg/L), which was supportive of, but not diagnostic for, pancreatitis; however, this diagnosis alone was not consistent with the dog’s presenting signs and clinical course. The final dog without a prompt response to doxycycline developed DIC and toxic epidermal necrolysis after only 7 days of doxycycline therapy; this dog was euthanized. Six of the nine initially responsive dogs developed signs consistent with CGA during the subsequent year (two of the owners could not be reached for follow-up). Each dog was treated empirically with an appropriate dose of doxycycline, and signs improved within 48 hours. Further serological testing was not available in these animals.

Discussion

One aim of this study was to describe the range of clinical presentations and responses to treatment in a series of dogs with serological evidence of exposure to A. phagocytophilum in Wisconsin. No significant differences were found between the number of females and males with antibody titers to A. phagocytophilum; this agreed with the findings from two recent studies but was different from a 76% female predominance previously reported in 1996.^3,20,21 Large-breed dogs (median weight 30.1 kg) predominated in this case series, which is consistent with other reports.²⁰ Age at presentation varied widely; notably, all dogs were >1 year of age, a finding also consistent with three other case series.^11,20,21

Fifty-eight percent of dogs (15 of 26) lived in the southern third of Wisconsin, and over half of these dogs resided in Dane County. This distribution may not reflect the actual geographic distribution of CGA in Wisconsin. The sample population was small, and the cases were taken from a tertiary referral veterinary medical center in Dane County in southern Wisconsin, which likely introduced a geographic selection bias into the study population. Previous authors have reported A. phagocytophilum infection in northwestern Wisconsin and northeastern Minnesota, but this case series highlights the risk for infection throughout Wisconsin—even in urban areas where the tick density is likely to be low.³

The most common clinical signs found in the 26 A. phagocytophilum-seropositive dogs in this study included lethargy, inappetence, and lameness—findings consistent with other studies.^3,6,13,15 About 50% of the dogs in this report were febrile. This percentage is lower than those reported for client-owned dogs with circulating morulae (88% to 93% febrile) and suggests that many of our dogs may have been presented to the referral center later in their clinical course.^3,20 In experimentally infected dogs, fever lasts <1 week and coincides with circulating A. phagocytophilum morulae.²²

Thrombocytopenia was observed in 56% of the dogs in this case series. Platelet counts were significantly lower in dogs that were also seropositive for antibodies to B. burgdorferi when compared with dogs seropositive for antibodies only to A. phagocytophilum. Others have also reported that dogs seropositive to both A. phagocytophilum and B. burgdorferi have more severe clinical signs overall compared with dogs having antibodies to A. phagocytophilum alone.⁶ Although B. burgdorferi infection is not typically associated with thrombocytopenia, low platelet counts have been reported in dogs with Borrelia-associated protein-losing nephropathy.²³ In a series of 17 dogs with acute CGA, all but one of six dogs with clinically significant thrombocytopenia (<100,000/μL) had either concurrent disease (lymphoma or systemic lupus erythematosus) or seropositivity to B. burgdorferi or E. canis, as well as to A. phagocytophilum.³ In a more recent study of 18 dogs with acute CGA with no comorbidities, the mean platelet count was low (76,000/μL).¹¹ Although some of these dogs had severe thrombocytopenia, modest thrombocytopenia appears to predominate in dogs with CGA.

The mechanism of the thrombocytopenia observed with A. phagocytophilum infection has not been established. Increased platelet destruction from antiplatelet antibodies has been proposed for E. canis infection and human granulocytic ehrlichiosis (HGE), and antiplatelet antibodies have been demonstrated recently in some dogs with A. phagocytophilum infection.^11,24–26 Bleeding was not observed in most of the dogs in our series. The two dogs with epistaxis had platelet counts of 138,000/μL and 85,000/μL, and epistaxis resolved during treatment with doxycycline and did not recur. Because spontaneous bleeding would not be expected at these platelet count levels, epistaxis may have been caused by an infection-induced vasculitis instead in these dogs.

Two dogs seropositive for antibodies to A. phagocytophilum alone were presented with cough. Cough was reported as a presenting complaint in three of eight dogs in one series²¹ but has not been reported in other studies.^3,11,20 In contrast, cough from interstitial pneumonia occurs in dogs with R. rickettsii infection and is commonly found in humans with HGE.²⁷ Nonproductive cough was reported in 70% of humans with confirmed HGE who resided in the upper midwest,²⁸ and atypical pneumonitis has been diagnosed in association with HGE in the United States and Europe.^29,30 Based on these observations, testing for A. phagocytophilum infection would be prudent in dogs presented with cough if more common etiologies are not found and if other clinical signs suggestive of CGA are present.

In this study, IMHA was diagnosed in three of 26 dogs. Two of these dogs were also seropositive for other organisms (E. risticii in one dog and both R. rickettsii and B. burgdorferi in the second dog). Infection with R. rickettsii and B. burgdorferi has not been associated with IMHA, and E. risticii infection is not associated experimentally with clinical disease in dogs.^23,31,32 The third dog with IMHA was seropositive to A. phagocytophilum only and had extensive diagnostic testing to exclude other triggers of the IMHA. Although IMHA has been reported in dogs seropositive to E. canis, IMHA has only been reported in one other dog with A. phagocytophilum infection.¹⁰ This previously reported dog had a progression similar to the dog in our case series; both developed DIC and were euthanized. None of the three dogs with IMHA in our series survived long enough to demonstrate a response to doxycycline. Although A. phagocytophilum infection was not definitively proven as the cause of IMHA, testing for A. phagocytophilum by PCR, serum antibody assays, and/or blood smear evaluation should be considered in dogs with IMHA in A. phagocytophilum-endemic areas.

Anaplasmosis is considered an acute infection that is promptly responsive to doxycycline; however, one of the 10 dogs seropositive for antibodies to A. phagocytophilum alone relapsed immediately after completing a single course of doxycycline therapy. This dog did respond to a second course of doxycycline. Four of five dogs that were seropositive to A. phagocytophilum alone and had adequate follow-up evaluations developed clinical signs consistent with A. phagocytophilum infection within the next year. Although recurrent A. phagocytophilum infection was not confirmed, all four dogs responded to doxycycline within 48 hours of starting therapy. A likely explanation for the return of clinical signs in these dogs was reexposure to A. phagocytophilum, as all four dogs lived in endemic areas, and repeated tick exposure was confirmed in each of the dogs. Recrudescence of infection is also possible, but this is a topic that requires more investigation.

Microscopic review of peripheral blood smears archived from 22 of the 26 dogs did not reveal granulocytic morulae in any dog. Acute and convalescent serological testing and A. phagocytophilum PCR were performed in only one of the 26 dogs. Therefore, an important limitation of this study was the use of unpaired A. phagocytophilum serological testing for inclusion of dogs, as the presence of antibodies to A. phagocytophilum does not prove active infection nor a causal relationship to clinical signs. Positive serological results can persist for several months in dogs naturally infected with A. phagocytophilum, and although antibodies are more common in dogs in Minnesota with clinical signs consistent with CGA, clinically normal dogs can also be seropositive for antibodies to A. phagocytophilum.^6,20,21 We attempted to address this limitation by focusing on clinically ill dogs that had follow-up after treatment with doxycycline; however, doxycycline can have nonspecific antiinflammatory effects (such as suppression of matrix metalloproteinases in arthritis) that may have interfered with our clinical interpretation of response to therapy.³³

Another noteworthy limitation of this study is that not all 26 dogs were tested for all other tick-borne organisms that may cause similar clinical signs. Twenty-four of the 26 dogs were screened for E. canis antibodies, but none of the dogs were tested for E. ewingii by PCR. Infection with E. ewingii can cause signs similar to those of CGA, and antigenic cross-reactivity exists between E. ewingii and E. canis.³⁴ Further, one study found that two dogs from Virginia and North Carolina with PCR-confirmed E. ewingii infections were seropositive to A. phagocytophilum using IFA testing.¹⁴ Ehrlichia ewingii infection appears to be uncommon in the upper midwest;⁶ therefore, infection with this organism seemed unlikely in the 26 dogs included in this study. Finally, seroconversion can occur before, during, or after the formation of granulocytic inclusions,²⁰ and dogs that had acute CGA may have been inadvertently excluded from this case series because of negative results at the time serological testing was done.

Conclusion

This retrospectively reviewed case series of 26 clinically ill, A. phagocytophilum-seropositive dogs supports previous findings that naturally infected dogs tend to be of large breeds and >1 year of age. Anaplasma phagocytophilum infection may be a trigger for IMHA in dogs, and cough and epistaxis may be presenting clinical signs. Dogs with antibodies to both A. phagocytophilum and B. burgdorferi have more severe thrombocytopenia than dogs with antibodies to A. phagocytophilum alone. Some dogs may have an apparent relapse of clinical signs after a therapeutic course of doxycycline; however, this observation requires further investigation.