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Typical and Atypical Manifestations of Anaplasmaphagocytophilum 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 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, arthrop- athy, lymphadenopathy, epistaxis, acute gastritis, cervical hyperpathia, and central nervous system dysfunction. Complete blood cell count abnormalities included thrombocytopenia, morulae in neutrophils, anemia, leukopenia, eosinopenia, lym- phopenia, 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) 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 America, and this trend is predicted to continue.3,4 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.1 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 ELISA enzyme-linked immunosorbent assay; IFA immunofluorescent assay; Pathogens Research Laboratory College of Veterinary Medicine, NCSU North Carolina State University; PCR polymerase chain reaction North Carolina State University, Raleigh, NC (P.D., E.B.); and IDEXX *P. Diniz's present affiliation is Small Animal Internal Medicine, Western Uni- Laboratories, Inc, Westbrook, ME (M.B., B.S., R.C.).
versity of Health Sciences College of Veterinary Medicine, Pomona, CA.
Correspondence: [email protected] (M.E.) 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 ofacute 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- 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 this assay does not cross-react with vaccinal antibodies and is considered to indicate active as opposed to previous infection.14 Materials and MethodsDogs 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 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 Canine DNA from a healthy dog was used as a PCR negative previously described.15 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.12 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.
triphosphate, and 1–5 mL of DNA template, according to the DNAconcentration determined for each sample (50–200 ng/reaction).
The GroEL and AnkA gene amplifications were performed as 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 ExaminationSixteen of 18 dogs (89%) were febrile (rectal temperature .
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 JAAHA 47:6 Nov/Dec 2011 Manifestations of Anaplasma phagocytophilum Infection Physical Examination Results Lameness, lethargy Labrador retriever Lameness, lethargy Lameness, lethargy Lameness, lethargy Cerebral dysfunction Lameness, lethargy Cervical hyperpathia Lameness, lethargy Labrador retriever Lameness, lethargy Polyarthropathy, acute Lameness, lethargy Polyarthropathy, mild 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 to680/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- 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).
Four dogs, three of which were A phagocytophilum seroreactive by SNAP 4Dx at the time of initial presentation, had follow-up 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, Enzyme-Linked Immunosorbent Assay (ELISA) Results Long-term follow up 28 mo later tested negative to all analytes 22 and 34 mo later tested negative to all analytes 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 In mouse studies, coinfection with A phagocytophilum and 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- 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 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 JAAHA 47:6 Nov/Dec 2011 Manifestations of Anaplasma phagocytophilum Infection A retrospective Swedish study did not show an association be- tween A phagocytophilum antibodies and the incidence of neu- Treatment and Outcomes rologic disease; however, because numerous tick exposed dogs in endemic regions become infected without developing clinically 6 mg/kg q 12 hr for 21 days Resolution in 2 days apparent illness, seroprevalence studies might have less utility 7 mg/kg q 12 hr for 28 days Resolution in 1 day than DNA-based testing modalities for establishing disease 7 mg/kg q 12 hr for 28 days Resolution in 4 days 7 mg/kg q 12 hr for 14 days Resolution in 2 days Dog 11 presented with a history of acute-onset lameness; 5 mg/kg q 12 hr for 14 days Resolution in 2 days 6 mg/kg q 12 hr for 14 days Resolution in 3 days cervical hyperpathia was the only abnormality identified during 7 mg/kg q 12 hr for 28 days Resolution in 5 days physical and neurologic examinations. The anatomic source of the 7 mg/kg q 12 hr for 28 days Resolution in 1 day, neck pain was unclear, but meningitis was considered a diagnostic with no furtherneurologic dysfunction possibility. As morulae were observed on the blood smear, cere- 8 mg/kg q 24 hr for 28 days Resolution in 2 days brospinal fluid was not sampled for analysis; thus, pain could have 8 mg/kg q 24 hr for 28 days Resolution in 6 days originated from the cervical musculature, cervical joints, or the 8 mg/kg q 12 hr for 28 days Resolution in 3 days intervertebral discs. The rapid resolution of pain and lameness 7 mg/kg q 24 hr for 30 days Resolution in 2 days after initiation of doxycycline, with no further problems or re- 8 mg/kg q 24 hr for 28 days Resolution in 3 days currence of disease reported, indicated that anaplasmosis could 8 mg/kg q 12 hr for 28 days Resolution in 5 days 6 mg/kg q 12 hr for 28 days Resolution in 1 day cause acute cervical pain in dogs.
7 mg/kg q 12 hr for 28 days Resolution in 2 days Bleeding problems were not reported with granulocytic an- 6 mg/kg q 12 hr for 28 days Resolution in 2 days aplasmosis but were described for other rickettsial infections in 8 mg/kg q 12 hr for 28 days Resolution in 3 days dogs, such as monocytic ehrlichiosis (E canis), cyclic thrombo- cytopenia (A platys), and Rocky Mountain Spotted Fever (Rickettsia ehrlichiosis, relatively few reports described central nervous system rickettsii).1,26,27 In addition to rickettsial organisms, other infec- disease resulting from A phagocytophilum infection.5 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 potentially life-threatening illnesses.24 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, 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 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 frame between illness of dog 6 and dog 15 made tick transmission doxycycline therapy, and no dog developed subsequent illness 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 a human infant who developed symptoms 9 days after birth.39 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 JAAHA 47:6 Nov/Dec 2011 Manifestations of Anaplasma phagocytophilum Infection clinical signs before the dog has a measureable antibody response.
DNeasy Blood & Tissue Kit; Qiagen, Inc, Valencia, CA Therefore, treatment decisions should not rely solely on antibody Applied Biosystems Thermocycler; Life Technologies Corp.,Carlsbad, CA testing, whether the results are negative or positive. The sero- negative dog might have been recently infected, whereas the se- ropositive dog might have been exposed months earlier and 1. McQuiston JH, McCall CL, Nicholson WL. Ehrlichiosis and related already eliminated the organism through innate immunity.
infections. J Am Vet Med Assoc 2003;223(12):1750–6.
Serology results must be considered in the context of physical 2. Bowman D, Little SE, Lorentzen L, et al. Prevalence and geographic examination, hematologic abnormalities, and potentially PCR distribution of Dirofilaria immitis, Borrelia burgdorferi, Ehrlichiacanis, and Anaplasma phagocytophilum in dogs in the United results. Long-term serological follow up using the in-clinic sero- States: results of a national clinic-based serologic survey. Vet Para- logic screening test was available for four dogs, of which three tested negative at subsequent testing intervals up to 3 yr. One dog 3. Madhav NK, Brownstein JS, Tsao JI, et al. A dispersal model for had a repeatable A phagocytophilum antibody response, which the range expansion of blacklegged tick (Acari: Ixodidae). J MedEntomol 2004;41(5):842–52.
might have been due to persistent antibody production or due to 4. Brownstein JS, Holford TR, Fish D. A climate-based model predicts re-exposure to A phagocytophilum infected ticks and an ama- the spatial distribution of the Lyme disease vector Ixodes scapularis nestic response after repeated transmission events. With the in the United States. Environ Health Perspect 2003;111(9):1152–7.
limited number of dogs and testing time points in this study, it 5. Greig B, Asanovich KM, Armstrong PJ, et al. Geographic, clinical, serologic, and molecular evidence of granulocytic ehrlichiosis, was not possible to draw conclusions as to how long after in- a likely zoonotic disease, in Minnesota and Wisconsin dogs. J Clin fection an individual dog would retain an A phagocytophilum antibody titer using the ELISA-based screening test. After nat- 6. Kohn B, Galke D, Beelitz P, et al. Clinical features of canine gran- ural infection in a group of 14 Swedish dogs, IFA titers decreased ulocytic anaplasmosis in 18 naturally infected dogs. J Vet InternMed 2008;22(6):1289–95.
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JAAHA 47:6 Nov/Dec 2011

Source: http://ws.westernu.edu/WesternU-News/docs/Eberts-2011-Typical-and-Atypical-Manifestations-of-Anaplasma-Infection-in-dogs.pdf