CASE SERIES Typical and Atypical Manifestations of Anaplasma phagocytophilum Infection in Dogs Matthew D. Eberts, DVM, Pedro Paulo Vissotto de Paiva Diniz, PhD, DVM*, Melissa J. Beall, DVM, Brett A. Stillman, PhD, Ramaswamy Chandrashekar, PhD, Edward B. Breitschwerdt, DVM ABSTRACT Eighteen clinically ill dogs, naturally infected with Anaplasma phagocytophilum, were examined at a veterinary practice in Baxter, Minnesota. A clinical examination, complete blood cell count, enzyme- linked immunosorbent assay (ELISA) for A phagocytophilum, Borrelia burgdorferi, and Ehrlichia canis antibodies and Diroﬁlaria immitis antigen, and a polymerase chain reaction test for A phagocytophilum DNA were obtained for all dogs. Physical examination ﬁndings included fever, arthropathy, lymphadenopathy, epistaxis, acute gastritis, cervical hyperpathia, and central nervous system dysfunction. Complete blood cell count abnormalities included thrombocytopenia, morulae in neutrophils, anemia, leukopenia, eosinopenia, lymphopenia, and monocytosis. Seroreactivity to A phagocytophilum was found in 61%, B burgdorferi antibodies in 17%, and D immitis antigen in 5% of the dogs. Fever, arthropathy, neurologic dysfunction, and epistaxis are clinical syndromes that can be associated with A phagocytophilum infection. Treatment with doxycycline resulted in rapid resolution of clinical signs in all dogs. (J Am Anim Hosp Assoc 2011; 47:e86–e94. DOI 10.5326/JAAHA-MS-5578) Introduction dogs in 1996.5 Granulocytic anaplasmosis can cause hematologic Anaplasma phagocytophilum is a gram-negative intracellular bac- abnormalities, most typically thrombocytopenia, leukopenia, and terium transmitted by Ixodes species ticks and is the causative anemia.5–8 The mechanism of thrombocytopenia is not fully un- agent of granulocytic anaplasmosis.1 In the United States, Ixodes derstood. but based on mouse experimental models and the scapularis is the vector for A phagocytophilum in the Northeast and presence of antiplatelet antibodies in naturally infected dogs, both Midwest, whereas Ixodes paciﬁcus vectors the organism on the consumptive- and immune-mediated mechanisms appear to be west coast.1 Surveys identiﬁed a wide distribution in seropreva- involved.6,9 Granulocytic intracytoplasmic morulae, which are lence among dogs in the United States, and high rates of exposure clusters of reproducing bacteria located in phagosomes, can be corresponded to the distribution of I scapularis and I paciﬁcus.2 In visualized during blood smear evaluation.10 Serum biochemical recent years, there were dynamic changes in the range of these tick abnormalities can include increased serum alkaline phosphatase vectors and their associated diseases. Climate change and changes activity, hypoalbuminemia, elevated serum alanine transferase, in wildlife distribution enabled expansion of I scapularis in North and elevated total bilirubin.5,6,8 3,4 America, and this trend is predicted to continue. Both enzyme-linked immunosorbent assay (ELISA) and in- A phagocytophilum has been documented to cause disease in direct ﬂuorescent antibody tests are commercially available, and a wide variety of species, including dogs, cats, horses, ruminants, documentation of seroconversion can be used to conﬁrm the and humans. Canine granulocytic anaplasmosis was ﬁrst de- diagnosis of granulocytic anaplasmosis. Experimental evidence scribed as an acute febrile disease in Minnesota and Wisconsin demonstrated that dogs might seroconvert within 10 days of From the Lakeland Veterinary Hospital, Baxter, MN (M.E.); Intracellular Pathogens Research Laboratory College of Veterinary Medicine, North Carolina State University, Raleigh, NC (P.D., E.B.); and IDEXX Laboratories, Inc, Westbrook, ME (M.B., B.S., R.C.). ELISA enzyme-linked immunosorbent assay; IFA immunofluorescent assay; 1 NCSU North Carolina State University; PCR polymerase chain reaction *P. Diniz’s present affiliation is Small Animal Internal Medicine, Western University of Health Sciences College of Veterinary Medicine, Pomona, CA. Correspondence: [email protected] (M.E.) e86 JAAHA | 47:6 Nov/Dec 2011 ª 2011 by American Animal Hospital Association Manifestations of Anaplasma phagocytophilum Infection exposure.11 Clinical illness usually occurs within 1–2 wk after tick NCSU). Cases were selected from 23 clinically ill dogs PCR tested transmission of A phagocytophilum; however, based on the high at NCSU and IDEXX Laboratories. Inclusion criteria for the cases seroprevalence in historically healthy dogs in endemic regions, selected for this study included molecular conﬁrmation of A most exposed dogs will not develop overt clinical disease. Due to phagocytophilum infection using PCR in conjunction with the the acute nature of this infection, approximately 40% of clinically availability of a medical history, physical examination ﬁndings, ill dogs will not produce a detectable level of A phagocytophilum a complete blood count, an in-clinic serologic assaya, and access to antibodies at the time of presentation when using current sero- the clinical response to doxycycline, as follow-up data. Eighteen logic tests.5–8 In contrast, A phagocytophilum polymerase chain dogs fulﬁlled these criteria (three dogs were excluded due to lack reaction (PCR) assays can be used to detect organism-speciﬁc of complete blood count at initial presentation, and two dogs were DNA sequences in the blood during the early stages of ill- excluded for lack of follow-up information). Anaplasmosis was ness. 6,11 With proper primer selection and validation, PCR testing diagnosed in all 18 cases between April and November 2005. provides a sensitive and speciﬁc modality for the diagnosis of acute anaplasmosis in animals and human patients, and a positive Complete Blood Counts PCR result is considered indicative of infection.10 However, it is Complete blood counts were performed at the time the dog pre- important to note that A phagocytophilum PCR can be positive in sented for diagnostic evaluation using an in-clinic laser ﬂow clinically normal dogs and periodically negative in actively cytometry automated unitb. Certiﬁed veterinary technicians vi- infected dogs, possibly due to variations in numbers of circulating sually examined a blood smear to evaluate cell morphology, organisms during infection.8,11,12 to perform differential cell counts, and to determine whether As a vector-borne infectious pathogen, A phagocytophilum morulae were present. can induce a variety of disease manifestations in dogs, which presents a challenge to practitioners attempting to accurately di- Serology agnose and determine effective treatment of their patients. Canine All dogs were tested at the time of presentation with a qualitative anaplasmosis is expanding geographically across North America. ELISA assay that detects Ehrlichia canis and Borrelia burgdorferi With increasing number of dogs exposed to this pathogen, natural antibodies, and Diroﬁlaria immitis antigenc. Retrospectively, all infection studies need to be disseminated to provide the veteri- dogs were also tested with a qualitative ELISA assay detecting nary community with a more complete deﬁnition of the spectrum E canis, B burgdorferi, and A phagocytophilum antibodies and of disease. It is important that practitioners be aware of potential D immitis antigena. The latter testing platform detects antibodies clinical signs and the tests available to diagnose anaplasmosis. The directed against a synthetic A phagocytophilum peptide from the purpose of this study is to describe clinical, hematologic, and major surface protein (p44/MSP2) and reportedly has 99.1% serologic ﬁndings and treatment outcomes for 18 dogs naturally sensitivity and 100% speciﬁcity compared with an immunoﬂuo- infected with A phagocytophilum in an I scapularis–endemic rescent assay (IFA) test.13 The B burgdorferi C6 peptide used in region. this assay does not cross-react with vaccinal antibodies and is considered to indicate active as opposed to previous infection.14 Materials and Methods Dogs Molecular Analysis Cases were retrospectively selected from a previous study involving PCR testing was performed either by personnel in the Vector Borne 731 dogs, 81% of which were purebred (Eberts, unpublished data). Disease Diagnostic Laboratory at NCSU or at IDEXX Laboratories These dogs were examined between July 2004 and January 2006 at in Westbrook, Maine. Eighteen ethylenediaminetetraacetic acid– a companion animal hospital located in Baxter, Minnesota, an area anticoagulated blood samples collected between April 2005 and endemic for I scapularis.12 Based on resources available at the time November of 2005 were processed for genomic DNA and analyzed of the study, retrospective PCR testing was performed at North by PCR to detect A phagocytophilum infection. NCSU tested Carolina State University (NSCU) on 273 dogs, of which 51 had a total of 14 samples, and IDEXX tested a total of 8 samples. clinical signs consistent with anaplasmosis and/or borreliosis.12 Of Four of the 18 samples were tested by both laboratories. DNA these 51 dogs, 19 tested PCR positive for A phagocytophilum was extracted from 200 mL of canine ethylenediaminetetra- DNA.12 Further retrospective PCR testing was performed at acetic acid whole-blood samples that were stored frozen at IDEXX Laboratories on eight additional clinical cases (four al- 2708C with a commercially available kitd. The ﬁnal eluted ready PCR tested at NCSU and four not previously tested at volume was 200 mL per sample. The DNA concentration was JAAHA.ORG e87 quantiﬁed by spectrophotometry, and the absence of PCR ampliﬁed PCR products were then evaluated on a 2% agarose gel inhibitors was demonstrated by the ampliﬁcation of a fragment stained with ethidium bromide. of the glyceraldehyde-3-phosphate dehydrogenase gene, as previously described.15 Canine DNA from a healthy dog was used as a PCR negative control. Plasmid clones of partial DNA sequences identical to the following accession numbers were also used as positive controls: North Carolina State University Protocol A platys 16S rRNA (AY821826), A platys GroEL (AF478129), PCR was performed using 16S rRNA oligonucleotide primers A phagocytophilum 16S rRNA (AF507941), A phagocytophilum AnkA that were designed to amplify all Anaplasma and Ehrlichia spe- (CP000235), and A phagocytophilum msp2 (AY151054). The DNA cies. Subsequently, the Anaplasma platys GroEL gene and the concentration of the puriﬁed plasmid was determined by re- A phagocytophilum AnkA gene were targeted as a secondary con- striction enzyme digestion and agarose gel electrophoresis (2%). ﬁrmation of the initial PCR result and to determine the infecting Plasmid clones were diluted 10-fold in puriﬁed dog DNA, ranging Anaplasma species (A phagocytophilum vs A platys). The oligonu- from 1 million to 1 plasmid/mL. The limit of detection observed cleotide primers were described elsewhere. The 16S rRNA gene in PCR ampliﬁcations was 16S rRNA ¼ 10 copies per reaction, ampliﬁcations were performed in a 25-mL ﬁnal volume reaction AnkA gene ¼ 25 copies per reaction, and GroEL gene ¼ 5 copies containing 13buffer with 2 mM magnesium chloride, 0.625 U of per reaction. The limit of detection was reached in 100% of tests. Takara Ex Taq DNA polymerase, 12.5 pmol of each primer, To prevent PCR amplicon contamination, sample extraction, re- 200 mM (each) of deoxyadenosine triphosphate, deoxythymidine action setup, PCR ampliﬁcation, and amplicon detection were 5’-triphosphate, deoxycytidine 5’-triphosphate, and deoxyguanosine performed in separated areas. 12 triphosphate, and 1–5 mL of DNA template, according to the DNA concentration determined for each sample (50–200 ng/reaction). Results The GroEL and AnkA gene ampliﬁcations were performed as Dogs previously described, with 7.5 pmol of each primer added in Fifteen of the 18 dogs (83%) were purebred, and the median age of each reaction. Conventional PCR was performed under the dogs was 6 yr (range, 3 mo to 14 yr). There were three intact following conditions: a single hot-start cycle at 958C for 2 min females, seven spayed females, three intact males, and ﬁve castrated followed by 55 cycles of denaturation at 948C for 15 sec, males. The most common presenting complaints were lethargy (13 annealing temperature 628C for 15 sec, and extension at 728C for dogs) and lameness (10 dogs), with circling, vomiting, and epi- 15 sec. Ampliﬁcation was completed by an additional cycle at staxis reported in 1 dog each. Median duration of clinical signs 728C for 1 min, and products were analyzed by 2% agarose gel before presentation was 3 days (range, 1–14 days) (Table 1). electrophoresis containing 0.2 mg of ethidium bromide/mL under ultraviolet light. Physical Examination Sixteen of 18 dogs (89%) were febrile (rectal temperature . IDEXX Protocol 39.28C), and the median temperature of febrile dogs was 40.28C A conventional PCR for A phagocytophilum was performed (range, 39.4–40.88C). Ten of 18 dogs (55%) had arthropathy, according to a published protocol and used the following msp2 which was deﬁned clinically as pain and swelling localized to primer pair: msp2–3f (59-CCAGCGTTTAGCAAGATAAGAG) joints on physical examination. Nine dogs (50%) had poly- and msp2–3r (59-GCCCAGTAACATCATAAGC).16 Ampliﬁcation arthropathy, and one dog (5%) had monoarthropathy involving was performed in a 25 mL volume reaction, containing a 13 the right radiocarpal joint. Lymphadenopathy, left cerebral dys- buffer mix of 1.5 mM magnesium chloride, 200 mM each of function, cervical hyperpathia, epistaxis, and acute gastritis were deoxyadenosine triphosphate, deoxythymidine 5’-triphosphate, found in one dog each (Table 1). deoxycytidine 5’-triphosphate, and deoxyguanosine triphosphate, 2.5 U of Taq DNA polymerase, 2 mL DNA template, and 0.5 mM Complete Blood Count of each the forward and reverse msp2 primer. The conventional Seventeen of 18 dogs (94%) were thrombocytopenic (normal range testing was performed in an Applied Biosystems Thermocyclere deﬁned as 175,000/mL to 500,000/mL), with platelet counts ranging with the following program: a single 4 min cycle at 948C followed from 40,000/mL to 120,000/mL (mean 70,941/mL). Morulae were by 40 cycles of denaturing at 948C for 30 sec, annealing at 558C identiﬁed in neutrophils during blood smear evaluation in 17 of for 30 sec, and extension at 728C for 1 min. Once the 40 cycles 18 dogs (94%). Twelve dogs (67%) had a nonregenerative normo- were complete, the samples were held for 5 min at 728C. The cytic normochromic anemia (normal range deﬁned as hematocrit e88 JAAHA | 47:6 Nov/Dec 2011 Manifestations of Anaplasma phagocytophilum Infection TABLE 1 Physical Examination Results Dog No. Breed Age Sex Examination date (2005) Complaint Duration of complaint (days) Temp.(8C) Examination interpretation 1 Cocker spaniel 5 mo Female April Lethargy 3 40.2 Fever 2 Springer spaniel 4 yr Female spayed April Lethargy 2 40.8 Fever 3 Basset hound 13 yr Male neutered April Lameness, lethargy 4 39.4 Polyarthropathy 4 Labrador retriever 2 yr Female spayed May Lameness, lethargy 3 40.8 Polyarthropathy 5 Mixed breed 2 yr Female spayed May Lameness, lethargy 14 40 Polyarthropathy 6 Border collie 1 yr Female May Lethargy 1 40.3 Fever 7 8 Mixed breed Airedale 10 yr 14 yr Female spayed Male neutered June June Lameness, lethargy Circling 3 1 40.7 38.2 Polyarthropathy Cerebral dysfunction 9 Shih tzu 6 yr Female spayed June Lameness 10 Golden retriever 5 yr Female spayed June Lameness, lethargy 14 39.9 Fever 3 38.9 Polyarthropathy 11 Cocker spaniel 8 yr Male neutered June Lameness 1 39.6 Cervical hyperpathia 12 Mixed breed 6 mo Male July Lameness, lethargy 2 40.4 Polyarthropathy 13 Shih tzu 6 yr Male neutered July Lethargy 7 40.4 Fever 14 Labrador retriever 2 yr Male neutered August Lameness, lethargy 2 39.9 Monoarthropathy 15 Border collie 3 mo Male September Vomiting 1 39.8 Polyarthropathy, acute gastritis 16 German shepherd 9 yr Female spayed October Lethargy 4 40.5 Fever 17 Golden retriever 12 yr Female October Lameness, lethargy 3 40.2 Polyarthropathy, mild lymphadenopathy 18 German shepherd 12 yr Male November Epistaxis 2 40.4 Polyarthropathy, epistaxis 37–55%), with hematocrits ranging from 25.7% to 36.9% (mean negative to A phagocytophilum (but morulae were observed in 32.5%). Ten dogs (55%) were leukopenic (normal range deﬁned neutrophils), and then tested negative to all analytes in June 2007 as 5,500–16,900/mL), with total white blood cell counts ranging from (22 mo later) and June 2008 (34 mo later). Dog 6 initially tested 3,670/mL to 5,420/mL (mean 4,432/mL). Eight dogs (50%) were positive for A phagocytophilum, and then tested positive for eosinopenic (normal eosinophil count deﬁned as 100–1,490/mL), A phagocytophilum in May 2008 (36 mo later). Dog 10 initially with eosinophil counts ranging from 20/mL to 80/mL (mean 55/mL), tested positive to A phagocytophilum but tested negative to all of which one dog had concurrent heartworm disease. Seven dogs analytes in June 2007 (25 mo later) and tested negative again in (39%) were lymphopenic (normal lymphocyte count deﬁned as June 2008 (37 mo later) (Table 2). 700–4,900/mL), with lymphocyte counts ranging from 200/mL to 680/mL (mean 512/mL), and one dog that presented for lameness Molecular Analysis and lethargy had a monocytosis (normal monocyte count de- As part of the retrospective evaluation, blood samples from all ﬁned as 100–1,400/mL) of 1,430/mL. This dog was concurrently dogs obtained at the time of presentation tested positive for infected with D immitis. A phagocytophilum DNA, targeting either the AnkA gene (n¼14) or the msp-2 gene (n¼8). Molecular evidence of infection cor- Serology related with the presence of morulae in all but one case where Of the 18 dogs in this study, A phagocytophilum antibodies morulae were not detected. Of the samples tested by both labora- were found in 11 (61%) dogs at the time of initial presentation; tories (n¼4), complete agreement was obtained. No molecular evi- B burgdorferi antibodies were found in 3 (17%); D. immitis antigens dence of A platys infection was detected in the 14 samples tested by were found in 1 (5%); and no dog was E canis seroreactive (Table 2). NCSU. Four dogs, three of which were A phagocytophilum seroreactive by SNAP 4Dx at the time of initial presentation, had follow-up Treatment testing using the in-clinic ELISA testa in subsequent years. Dog Treatment with doxycycline at a dose ranging from 6 to 8 mg/kg PO q 1 initially tested positive for A phagocytophilum but tested negative 12–24 hr for 14–30 days resulted in a positive therapeutic response for all analytes in August 2007 (28 mo later). Dog 2 initially tested in all dogs. Three dogs were treated for 14 days, 1 dog for 21 days, JAAHA.ORG e89 TABLE 2 Enzyme-Linked Immunosorbent Assay (ELISA) Results Anaplasma phagocytophilum antibodies Borrelia burgdorferi antibodies Ehrlichia canis antibodies Dirofilaria immitis antigen 1 Yes No No No 28 mo later tested negative to all analytes 2 No No No No 22 and 34 mo later tested negative to all analytes 3 Yes Yes No Yes 4 5 No Yes No No No No No No 6 Yes No No No 7 No Yes No No Dog no. 8 Yes No No No 9 No No No No 10 Yes No No No 11 Yes No No No 12 13 No No No No No No No No 14 Yes Yes No No 15 Yes No No No 16 Yes No No No 17 No No No No 18 Yes No No No 11 (61%) 3 (17%) 0 (0%) 1 (5%) No. positive (%) Long-term follow up 36 mo later tested positive to Anaplasma 25 and 37 mo later tested negative to all analytes and 14 dogs for 28–30 days. Treatment duration did not appear to A phagocytophilum and B burgdorferi.12 The timing of the inﬂuence outcome, as disease manifestations resolved within 24 hr B burgdorferi infection could not be determined in these dogs, in three dogs, including the dog with central nervous system but it might represent a prior transmission event, because up to dysfunction, 48 hr in seven dogs, 72 hr in four dogs, and 4–6 days 6 wk is generally required for the dog to develop a detectable in the remaining four dogs (Table 3). antibody response after tick attachment.21 Discussion B burgdorferi alters the host immune response, which can lead For the dogs described in this study, fever accompanied by ar- to increased severity of Lyme arthritis.22,23 In the original serosurvey, thropathy was the most common clinical presentation associated coinfected dogs were more likely to have lameness, joint pain, with A phagocytophilum infection. On physical examination, 10 of and joint effusion than dogs with single infections.12 In this the 18 dogs had joint pain and effusion. Lyme disease, which is study, 3 of the 18 dogs were coinfected with B burgdorferi (dogs caused by B burgdorferi and transmitted by the same Ixodes ticks, 3, 7, and 14) (Table 2). All three presented with arthropathy also often manifests as a febrile arthropathy.14 One challenge (Table 1), and the severity of clinical signs did not appear to be when trying to deﬁne the clinical disease that is associated with worse than the other dogs with arthropathy. The coinfected dogs canine anaplasmosis is concurrent or sequential transmission of were thrombocytopenic; however, the degree of thrombocyto- In mouse studies, coinfection with A phagocytophilum and A phagocytophilum and B burgdorferi. Although it is clear that penia was not more severe than dogs without borreliosis. When dogs in nature can experience frequent and repeated tick looking at response to doxycycline, 4 of the 18 dogs in this study infestations, only a small percentage of infected ticks contain took .3 days for clinical signs to completely resolve (Table 3). It these two pathogens, yet individual ticks may be infected with is interesting to note that, of those four dogs, three were coin- one or both organisms. For example, depending on location, fected with B burgdorferi. The limited number of coinfected dogs studies from the Midwest identify B burgdorferi in 16.5–57% and makes it difﬁcult to determine whether this has true clinical A phagocytophilum in 3.8–14% of collected I scapularis.17–20 In the relevance. study from which these dogs were selected, disease manifestations Although neurologic signs were reported for other rickettsial were more often observed in dogs that were coinfected with infections in the dog, such as Rocky Mountain Spotted Fever and e90 JAAHA | 47:6 Nov/Dec 2011 Manifestations of Anaplasma phagocytophilum Infection A retrospective Swedish study did not show an association be- TABLE 3 tween A phagocytophilum antibodies and the incidence of neu- Treatment and Outcomes Dog No. rologic disease; however, because numerous tick exposed dogs in Doxycycline Outcome endemic regions become infected without developing clinically 1 2 6 mg/kg q 12 hr for 21 days 7 mg/kg q 12 hr for 28 days Resolution in 2 days Resolution in 1 day apparent illness, seroprevalence studies might have less utility 3 7 mg/kg q 12 hr for 28 days Resolution in 4 days 4 7 mg/kg q 12 hr for 14 days Resolution in 2 days 5 5 mg/kg q 12 hr for 14 days Resolution in 2 days 6 6 mg/kg q 12 hr for 14 days Resolution in 3 days cervical hyperpathia was the only abnormality identiﬁed during 7 7 mg/kg q 12 hr for 28 days Resolution in 5 days physical and neurologic examinations. The anatomic source of the 8 7 mg/kg q 12 hr for 28 days Resolution in 1 day, with no further neurologic dysfunction neck pain was unclear, but meningitis was considered a diagnostic than DNA-based testing modalities for establishing disease associations.25 Dog 11 presented with a history of acute-onset lameness; possibility. As morulae were observed on the blood smear, cere- 9 8 mg/kg q 24 hr for 28 days Resolution in 2 days 8 mg/kg q 24 hr for 28 days brospinal ﬂuid was not sampled for analysis; thus, pain could have 10 Resolution in 6 days 8 mg/kg q 12 hr for 28 days originated from the cervical musculature, cervical joints, or the 11 Resolution in 3 days 12 13 7 mg/kg q 24 hr for 30 days 8 mg/kg q 24 hr for 28 days Resolution in 2 days Resolution in 3 days 14 8 mg/kg q 12 hr for 28 days Resolution in 5 days currence of disease reported, indicated that anaplasmosis could 15 6 mg/kg q 12 hr for 28 days Resolution in 1 day cause acute cervical pain in dogs. 16 7 mg/kg q 12 hr for 28 days Resolution in 2 days Bleeding problems were not reported with granulocytic an- 17 6 mg/kg q 12 hr for 28 days Resolution in 2 days 18 8 mg/kg q 12 hr for 28 days aplasmosis but were described for other rickettsial infections in Resolution in 3 days intervertebral discs. The rapid resolution of pain and lameness after initiation of doxycycline, with no further problems or re- dogs, such as monocytic ehrlichiosis (E canis), cyclic thrombocytopenia (A platys), and Rocky Mountain Spotted Fever (Rickettsia ehrlichiosis, relatively few reports described central nervous system 5 rickettsii).1,26,27 In addition to rickettsial organisms, other infec- disease resulting from A phagocytophilum infection. Dog 8 tions were associated with canine epistaxis, including aspergil- presented with acute onset altered mentation and circling to the losis, bartonellosis, and leshmaniaisis.28–30 Dog 18 presented left. Cranial nerve examination, peripheral reﬂexes, postural with epistaxis, accompanied by fever, joint pain, and thrombo- reactions, and proprioception were normal. Neurologic exami- cytopenia. The platelet count was 101,000/mL, which would nation was consistent with left cerebral dysfunction. It is in- suggest that factors other than thrombocytopenia were associated teresting to note that this dog was geriatric (14 yr old) and did with spontaneous bleeding in this dog. Although the mecha- not present with fever or evidence of joint pain or effusion. nism of epistaxis was not determined, rapid and sustained res- Although fatal illness due to anaplasmosis has not been reported olution of bleeding occurred after initiation of doxycycline in dogs, elderly humans are more likely to develop severe and therapy. Further diagnostic testing was not performed. In the 24 potentially life-threatening illnesses. The complete blood count dog, it appears that polymicrobial infections might be an impor- ﬁndings were consistent with anaplasmosis, and morulae were tant factor to consider diagnostically when examining infectious easily identiﬁed on the blood smear. Despite the advanced age causes of epistaxis.24,28 Because of the atypical presentation in of this dog and the neurologic presentation, rapid resolution of this dog, it was an intriguing possibility that multiple vector clinical signs occurred within 24 hr after the initiation of borne infections might have been involved, but in the absence doxycycline therapy. Further diagnostics, such as brain imaging of further diagnostic testing, this conclusion remained purely and cerebrospinal ﬂuid collection, were not performed; thus, speculative. other neurologic disorders could not be deﬁnitively ruled out. The medical history describing a rapid onset illness, in However, the dog had no history of neurologic disease, and no conjunction with the temporal relationship of the clinical pre- further neurologic problems were reported during the remaining sentation with I. scapularis activity in central Minnesota, sup- months of his life. Dog 8 was euthanatized 4 mo after presentation ported an acute disease process rather than acute decompensation for quality-of-life issues associated with progressive osteoarthritis, after chronic A phagocytophilum infection. Dogs most often pre- a pre-existing condition. Although neurologic dysfunction is sented in the spring and fall with clinical disease at a time that a seemingly uncommon manifestation of anaplasmosis, rapid coincided with the highest numbers of I scapularis in the envi- diagnosis and effective treatment can elicit a favorable outcome. ronment.31 Of the 273 dogs tested by PCR in the original JAAHA.ORG e91 serosurvey, no dog tested A phagocytophilum positive during the cow gave birth to an infected calf, which had clinical signs con- months of December, January, February, and March.12 sistent with anaplasmosis 13 days after birth.41 The 14 wk time All of the dogs in this study responded clinically to doxycycline therapy, and no dog developed subsequent illness frame between illness of dog 6 and dog 15 made tick transmission more likely than perinatal transmission. consistent with granulocytic anaplasmosis. To date, there have The complete blood count is a useful diagnostic test for been no clinical reports that document chronic infection with granulocytic anaplasmosis. Thrombocytopenia is a common he- A phagocytophilum; however, there was experimental evidence that matologic abnormality and evaluation of stained blood smears supported this possibility. Two experimental studies demonstrated facilitates visualization of granulocyte morulae in acutely infected presence of A phagocytophilum DNA months after initial infection dogs. For veterinary clinics that obtain in-house automated and even after doxycycline therapy.32,33 Importantly, notable complete blood counts, having a trained technician visually clinical signs other than thrombocytopenia did not occur after evaluate blood smears is critically important for the diagnosis of the acute phase of infection, and viable organisms could not be anaplasmosis, as well as other tick-borne infections, including transmitted by blood transfusion from a PCR positive dog to babesiosis, cytauxzoonosis, ehrlichiosis, and hepatzoonosis.42 a naïve dog. In addition to chronic infection, the possibility of Morulae provide rapid diagnostic information but are not present reinfection exists for dogs in highly endemic areas where dogs in every infection. Another problem with morulae is that based on may be re-exposed to anaplasmosis over months and years. There microscopy alone A phagocytophilum morulae are indistinguish- is evidence to support A phagocytophilum strain variation in able from E ewingii morulae.10 Dependent on the skill level and various regions of the United States and internationally. 34,35 There experience of the technician, other granulocyte changes or arti- are no data to deﬁne whether a dog can be reinfected with ho- facts (e.g., other cytoplasmic structures or stain precipitate) have mologous or heterologous A phagocytophilum strains. As in pre- the potential to be misdiagnosed as A phagocytophilum inclusions. vious studies, doxycycline was an effective treatment of canine In cases where no morulae are seen, PCR testing can facilitate anaplasmosis.5–8 The ideal duration of therapy has not been an accurate diagnosis, which offers the advantage of differentiating established, although a 28 day course of doxycycline at 10 mg/kg q between other infectious and noninfectious diseases that have 24 hr PO has been recommended.36 In human medicine, current similar hematologic and clinical presentations. In this study, a PCR recommendations call for a 10 day treatment of doxycycline for positive test was part of the inclusion criteria to insure that the dogs granulocytic anaplasmosis.37 In this case series, 3 dogs were had active infection at the time of presentation. This introduced treated for 14 days, 1 dog for 21 days, 13 dogs for 28 days, and 1 a bias, because in the original study from which the cases were dog for 30 days. All dogs responded to treatment in a similar selected, not all suspected cases of anaplasmosis were PCR positive. manner. In experimentally infected dogs, A phagocytophilum DNA For example, there were 16 morulae positive dogs tested by PCR persisted after doxycycline treatment of 14 and 28 days.33,38 Be- and 14 were positive.12 The two PCR-negative, morulae-positive cause dogs were not followed with sequential PCR testing, chronic dogs might have represented false-negative PCR results or the or reinfection in asymptomatic recovered dogs in this study could morulae might have been misdiagnosed. Another potential pitfall not be determined. is that clinically normal dogs can test A phagocytophilum PCR Three puppies, dog 1 (5 mo of age), dog 12 (6 mo of age), and positive.8,12 In the original serosurvey, seven asymptomatic dog 15 (3 mo of age) were described in this case series. Dogs 1, 12, dogs were PCR positive, four of which were also seropositive.12 and 15 were treated with doxycycline for 21, 30, and 28 days, These dogs likely represented asymptomatic acute infections; respectively, with no evidence of tooth discoloration. It was in- however, in the four seropositive PCR positive dogs, asymptomatic teresting to note that dog 15, an offspring of dog 6, was born 2 wk chronic infection was a possibility. These data suggested that an before the onset of illness in the dam. None of the other puppies in ill PCR positive dog might have clinical disease unrelated to that litter had problems during nursing, weaning, or when placed A phagocytophilum infection. Failure to achieve a prompt treatment in new homes. Dog 6 remained in the same household as the dam response to doxycycline would tend to rule out canine anaplas- and developed clinical disease 14 wk after the dam’s illness. mosis or could suggest coinfection with organisms that were not Perinatal transmission of A phagocytophilum was reported in doxycycline responsive. 39 a human infant who developed symptoms 9 days after birth. A Seven dogs in this study had negative A phagocytophilum recent study involving a bitch naturally infected just before serologic test results at the time of diagnosis, which was not whelping failed to demonstrate perinatal transmission to the surprising because acute infection with vector-borne organisms, puppies.40 In a bovine experimental infection study, an infected including Anaplasma, Ehrlichia, and Rickettsia spp., can cause e92 JAAHA | 47:6 Nov/Dec 2011 Manifestations of Anaplasma phagocytophilum Infection clinical signs before the dog has a measureable antibody response. Therefore, treatment decisions should not rely solely on antibody testing, whether the results are negative or positive. The seronegative dog might have been recently infected, whereas the seropositive dog might have been exposed months earlier and already eliminated the organism through innate immunity. Serology results must be considered in the context of physical examination, hematologic abnormalities, and potentially PCR results. Long-term serological follow up using the in-clinic serologic screening test was available for four dogs, of which three tested negative at subsequent testing intervals up to 3 yr. One dog had a repeatable A phagocytophilum antibody response, which might have been due to persistent antibody production or due to re-exposure to A phagocytophilum infected ticks and an amanestic response after repeated transmission events. With the limited number of dogs and testing time points in this study, it was not possible to draw conclusions as to how long after infection an individual dog would retain an A phagocytophilum antibody titer using the ELISA-based screening test. After natural infection in a group of 14 Swedish dogs, IFA titers decreased below the diagnostic cutoff in some dogs by 4–12 mo after infection; however, variations in immune response in conjunction with the possibility of reinfection made predictions for clinical practice problematic.7 Conclusion As diagnostic capabilities available to practicing veterinarians continue to increase, the spectrum of clinical disease associated with canine granulocytic anaplasmosis will be further reﬁned. Although an acute febrile illness accompanied by lameness or joint effusion is seemingly the most typical presentation of canine anaplasmosis, clinicians should be aware of other infrequent or atypical presentations. Neurologic disease, lymphadenopathy, vomiting, and epistaxis were the predominant abnormalities in some dogs in this study. Prompt treatment was associated with a rapid clinical response and the long-term prognosis appeared to be excellent. In cases where anaplasmosis is suspected but the dog does not respond to doxycycline, other disease processes should be considered. The costs of molecular analysis and retrospective serologic testing were paid for by IDEXX Laboratories. FOOTNOTES a SNAP 4Dx Test; IDEXX Laboratories, Inc, Westbrook, ME b LaserCyte Hematology Analyzer; IDEXX Laboratories, Inc, Westbrook, ME c SNAP 3Dx Test; IDEXX Laboratories, Inc, Westbrook, ME d e DNeasy Blood & Tissue Kit; Qiagen, Inc, Valencia, CA Applied Biosystems Thermocycler; Life Technologies Corp., Carlsbad, CA REFERENCES 1. McQuiston JH, McCall CL, Nicholson WL. Ehrlichiosis and related infections. J Am Vet Med Assoc 2003;223(12):1750–6. 2. Bowman D, Little SE, Lorentzen L, et al. Prevalence and geographic distribution of Diroﬁlaria immitis, Borrelia burgdorferi, Ehrlichia canis, and Anaplasma phagocytophilum in dogs in the United States: results of a national clinic-based serologic survey. Vet Parasitol 2009;160(1-2):138–48. 3. Madhav NK, Brownstein JS, Tsao JI, et al. A dispersal model for the range expansion of blacklegged tick (Acari: Ixodidae). J Med Entomol 2004;41(5):842–52. 4. Brownstein JS, Holford TR, Fish D. A climate-based model predicts the spatial distribution of the Lyme disease vector Ixodes scapularis in the United States. Environ Health Perspect 2003;111(9):1152–7. 5. Greig B, Asanovich KM, Armstrong PJ, et al. Geographic, clinical, serologic, and molecular evidence of granulocytic ehrlichiosis, a likely zoonotic disease, in Minnesota and Wisconsin dogs. J Clin Microbiol 1996;34(1):44–8. 6. Kohn B, Galke D, Beelitz P, et al. Clinical features of canine granulocytic anaplasmosis in 18 naturally infected dogs. J Vet Intern Med 2008;22(6):1289–95. 7. Egenvall AE, Hedhammar AA, Bjöersdorff AI. Clinical features and serology of 14 dogs affected by granulocytic ehrlichiosis in Sweden. Vet Rec 1997;140(9):222–6. 8. Granick JL, Armstrong PJ, Bender JB. Anaplasma phagocytophilum infection in dogs: 34 cases (2000-2007). J Am Vet Med Assoc 2009; 234(12):1559–65. 9. Borjesson DL, Simon SI, Tablin F, et al. Thrombocytopenia in a mouse model of human granulocytic ehrlichiosis. J Infect Dis 2001; 184(11):1475–9. 10. Greig B, Armstrong PJ. Canine granulocytotropic anaplasmosis (A. phagocytophilum infection). In: Greene CE, ed. Infectious diseases of the dog and cat. 3rd ed. St. Louis: Saunders; 2006: 219–24. 11. Egenvall A, Bjöersdorff A, Lilliehöök I, et al. Early manifestations of granulocytic ehrlichiosis in dogs inoculated experimentally with a Swedish Ehrlichia species isolate. Vet Rec 1998;143(15):412–7. 12. Beall MJ, Chandrashekar R, Eberts MD, et al. Serological and molecular prevalence of Borrelia burgdorferi, Anaplasma phagocytophilum, and Ehrlichia species in dogs from Minnesota. Vector Borne Zoonotic Dis 2008;8(4):455–64. 13. Chandrashekar R, Mainville C, Daniluk D, et al. Performance of an in-clinic test SNAP 4Dx, for the detection of antibodies to canine granulocytic infection, Anaplasma phagocytophilum (abstract). J Vet Intern Med 2007;21:626. 14. Greene C, Straubinger R. Borreliosis. In: Greene CE, ed. Infectious diseases of the dog and cat. 3rd ed. St. Louis: Saunders; 2006:417–35. 15. Birkenheuer AJ, Levy MG, Breitschwerdt EB. Development and evaluation of a seminested PCR for detection and differentiation of Babesia gibsoni (Asian genotype) and B. canis DNA in canine blood samples. J Clin Microbiol 2003;41(9):4172–7. 16. Massung RF, Slater K, Owens JH, et al. Nested PCR assay for detection of granulocytic ehrlichiae. J Clin Microbiol 1998;36(4): 1090–5. JAAHA.ORG e93 17. Layﬁeld D, Guilfoile P. The prevalence of Borrelia burgdorﬁeri (Spirochaetales: spirochaetaceae) and the agent of human granulocytic ehrlichiosis (Rickettsiaceae: Ehrlichieae) in Ixodes scapularis (Acari:Ixodidae) collected during 1998 and 1999 from Minnesota. J Med Entomol 2002;39(1):218–20. 18. Piesman J, Clark KL, Dolan MC, et al. Geographic survey of vector ticks (Ixodes scapularis and Ixodes paciﬁcus) for infection with the Lyme disease spirochete, Borrelia burgdorferi. J Vector Ecol 1999;24 (1):91–8. 19. Michalski M, Rosenﬁeld C, Erickson M, et al. Anaplasma phagocytophilum in central and western Wisconsin: a molecular survey. Parasitol Res 2006;99(6):694–9. 20. Steiner FE, Pinger RR, Vann CN, et al. Infection and co-infection rates of Anaplasma phagocytophilum variants, Babesia spp., Borrelia burgdorferi, and the rickettsial endosymbiont in Ixodes scapularis (Acari: Ixodidae) from sites in Indiana, Maine, Pennsylvania, and Wisconsin. J Med Entomol 2008;45(2):289–97. 21. Liang FT, Jacobson RH, Straubinger RK, et al. Characterization of a Borrelia burgdorferi VlsE invariable region useful in canine Lyme disease serodiagnosis by enzyme-linked immunosorbent assay. J Clin Microbiol 2000;38(11):4160–6. 22. Thomas V, Anguita J, Barthold SW, et al. Coinfection with Borrelia burgdorferi and the agent of human granulocytic ehrlichiosis alters murine immune responses, pathogen burden, and severity of Lyme arthritis. Infect Immun 2001;69(5):3359–71. 23. Zeidner NS, Dolan MC, Massung R, et al. Coinfection with Borrelia burgdorferi and the agent of human granulocytic ehrlichiosis suppresses IL-2 and IFN g production and promotes an IL-4 response in C3H/HeJ mice. Parasite Immunol 2000;22(11):581–8. 24. Bakken JS, Dumler JS. Clinical diagnosis and treatment of human granulocytotropic anaplasmosis. Ann N Y Acad Sci 2006;1078: 236–47. 25. Jäderlund KH, Egenvall A, Bergström K, et al. Seroprevalence of Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum in dogs with neurological signs. Vet Rec 2007;160(24):825–31. 26. Wilson J. Ehrlichia platys in a Michigan dog. J Am Anim Hosp Assoc 1992;28:381–3. 27. Greene C, Breitschwerdt E. Rocky mountain spotted fever, murine typhuslike disease, rickettsialpox, typhus, and Q fever. In: Greene CE, ed. Infectious diseases of the dog and cat. 3rd ed. St. Louis: Saunders; 2006:232–45. 28. Breitschwerdt EB, Hegarty BC, Maggi R, et al. Bartonella species as a potential cause of epistaxis in dogs. J Clin Microbiol 2005;43(5): 2529–33. 29. Jüttner C, Rodríguez Sánchez M, Rollán Landeras E, et al. Evaluation of the potential causes of epistaxis in dogs with natural visceral leishmaniasis. Vet Rec 2001;149(6):176–9. e94 JAAHA | 47:6 Nov/Dec 2011 30. Mylonakis ME, Saridomichelakis MN, Lazaridis V, et al. A retrospective study of 61 cases of spontaneous canine epistaxis (1998 to 2001). J Small Anim Pract 2008;49(4):191–6. 31. Daniels TJ, Falco RC, Curran KL, et al. Timing of Ixodes scapularis (Acari: Ixodidae) oviposition and larval activity in southern New York. J Med Entomol 1996;33(1):140–7. 32. Egenvall A, Lilliehöök I, Bjöersdorff A, et al. Detection of granulocytic Ehrlichia species DNA by PCR in persistently infected dogs. Vet Rec 2000;146(7):186–90. 33. Alleman A, Chandrashekar R, Beall M, et al. Experimental inoculation of dogs with a human isolate (Ny18) of Anaplasma phagocytophilum and demonstration of persistent infection following doxycycline therapy (abstract). J Vet Intern Med 2006; 20:763. 34. Barbet AF, Lundgren AM, Alleman AR, et al. Structure of the expression site reveals global diversity in MSP2 (P44) variants in Anaplasma phagocytophilum. Infect Immun 2006;74(11): 6429–37. 35. Foley J, Nieto NC, Madigan J, et al. Possible differential host tropism in Anaplasma phagocytophilum strains in the Western United States. Ann N Y Acad Sci 2008;1149:94–7. 36. Neer TM, Breitschwerdt EB, Greene RT, et al; American College of Veterinary Internal Medicine. Consensus statement on ehrlichial disease of small animals from the infectious disease study group of the ACVIM. J Vet Intern Med 2002;16(3):309–15. 37. Wormser GP, Dattwyler RJ, Shapiro ED, et al. The clinical assessment, treatment, and prevention of lyme disease, human granulocytic anaplasmosis, and babesiosis: clinical practice guidelines by the Infectious Diseases Society of America. Clin Infect Dis 2006;43(9): 1089–134. 38. Alleman A, Wamsley H, Abbott J, et al. Experimental Anaplasma phagocytophilum infection of dogs by intravenous inoculation of human and canine isolates and treatment with doxycycline (abstract). Vet Pathol 2007;44:19. 39. Dhand A, Nadelman RB, Aguero-Rosenfeld M, et al. Human granulocytic anaplasmosis during pregnancy: case series and literature review. Clin Infect Dis 2007;45(5):589–93. 40. Plier ML, Breitschwerdt EB, Hegarty BC, et al. Lack of evidence for perinatal transmission of canine granulocytic anaplasmosis from a bitch to her offspring. J Am Anim Hosp Assoc 2009;45(5): 232–8. 41. Pusterla N, Braun U, Wolfensberger C, et al. Intrauterine infection with Ehrlichia phagocytophila in a cow. Vet Rec 1997;141(4): 101–2. 42. Walker D. Peripheral blood smears. In: Cowell R, Tyler R, Meinkoth J, et al, eds. Diagnostic cytology and hematology of the dog and cat. 3rd ed. St. Louis: Mosby; 2007:254–83.
© Copyright 2018