JFM0010.1177/1098612X15571148Journal of Feline Medicine and SurgerySavidge et al r571148esearch-article2015

Original Article

Journal of Feline Medicine and Surgery 7–­1 Anaplasma phagocytophilum © ISFM and AAFP 2015 Reprints and permissions: infection of domestic cats: 16 cases sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/1098612X15571148 from the northeastern USA jfms.com

Christine Savidge1, Patty Ewing2, Jan Andrews3, David Aucoin4, Michael R Lappin5 and Scott Moroff6

Abstract Objectives Anaplasma phagocytophilum is an Ixodes species-transmitted rickettsial organism that is occasionally associated with clinical abnormalities in humans, ruminants, horses, dogs and cats. While serological evidence of A phagocytophilum exposure is common in cats in Ixodes species endemic areas, reports of clinical feline are few. The objective of this study was to describe the clinical and laboratory abnormalities and treatment responses in 16 cats with A phagocytophilum DNA amplified from blood. Methods Commercial laboratory electronic records were searched to find cats that had A phagocytophilum DNA amplified from their blood. Once cases were identified, the primary care veterinarian was interviewed and the medical records were reviewed. Results The cats ranged in age from 4 months to 13 years (mean 4.1 years, median 2 years). All cats lived in Ixodes scapularis endemic areas and had potential for exposure. All cats were lethargic, 15 (94%) had elevated body temperature (>39.4°C) and 14 were anorexic on initial physical examination. Other less common clinical findings included hepatosplenomegaly, ataxia, and elevation of the nictitating membranes. Blood from 11 cats was evaluated by complete blood cell count; abnormalities included lymphopenia in seven (64%) cats, in seven (64%), morulae in neutrophils of three (27%), neutropenia in three (27%) and leukopenia in two (18%). Treatment responses were reported for 14 cats, and the clinical abnormalities in these cats resolved when was administered. Conclusions and relevance This is the first published report describing A phagocytophilum morulae in neutrophils of naturally infected North American cats with infection confirmed by PCR. A phagocytophilum infection should be considered in cats evaluated for lethargy, anorexia and fever living in Ixodes species endemic areas.

Accepted: 12 January 2015

Introduction Anaplasma phagocytophilum is a rickettsial organism that 1Department of Companion Animals, Atlantic Veterinary College, causes granulocytic anaplasmosis in cats, dogs, horses, Charlottetown, PE, Canada 2Angell Animal Medical Center, Boston, MA, USA ruminants and humans. The organism was first 3Antech Diagnostics, Cary, NC, USA described in 1932 in Scottish sheep,1 and then reported 4Antech Diagnostics, Santa Monica, CA, USA to affect horses, dogs, cats, cattle, camelids and humans. 5Department of Clinical Sciences, Colorado State University, The worldwide distribution of A phagocytophilum fol- Ft Collins, CO, USA 6Antech Diagnostics, Lake Success, NY, USA lows the geographic distribution of the primary vector, Ixodes species.2 In North America, the organism is trans- This research was presented, in part, as an abstract at the Annual Forum of the American College of Veterinary Internal Medicine in mitted by Ixodes scapularis in the Northeast and Denver, Colorado, June 2011 Midwest, and by Ixodes pacificus in the West.3 Infections are highest in the late spring and autumn when both Corresponding author: nymph and adult ticks are most mobile.4 Transmission Christine Savidge DVM, DACVIM, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, to mammals occurs within 24–48 h of tick Charlottetown, PE, C1A 4P3, Canada attachment.5,6 Email: [email protected]

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Once transmitted, A phagocytophilum infects circulat- synthetic targets contained exact sequence matches to ing neutrophils forming intracellular inclusions (moru- the primer sequences at the exact sequence distances lae), which can be observed via light microscopy on a found in the intended reference target sequences. An Romanowsky or Wright-Giemsa-stained blood smear. In internal validation study was performed and assays North America, morulae have been identified in the neu- were determined to be sensitive to 20 copies of their syn- trophils of naturally exposed dogs,4 horses,7 humans8 thetic DNA targets. and experimentally infected cats.9 In Europe, morulae of The attending veterinarians were then contacted by A phagocytophilum have been described in ruminants,10 telephone to obtain clinical information from the case cats,11 horses12 and dogs.13 records. Data abstracted from the medical record There are multiple papers describing anaplasmosis in included history (duration of signs, outdoor activity, tick dogs.4,14–16 The first reports of granulocytic anaplasmosis exposure, use of acaricides and geographic location), in naturally infected cats primarily reported non-specific physical examination findings, clinicopathologic data, clinical signs of lethargy, anorexia, fever and dehydra- date of the PCR test, diagnostic imaging at the time of tion.11,17–21 Clinicopathologic abnormalities in infected diagnosis, treatments administered and response to cats often include neutrophilia, lymphopenia and hyper- treatment. Clinicopathologic data came from multiple glycemia with or without thrombocytopenia. Diagnosis diagnostic laboratories. Numerical data were evaluated of A phagocytophilum can be made based on clinical sus- using manually calculated standard descriptive statistics picion, a history of exposure to Ixodes species, identifica- (range, mean, frequency). tion of morulae within neutrophils, positive serologic results, PCR results and response to treatment.19 Results Naturally infected cats produce serologic antibodies to A Between May 2009 and May 2011, 4334 blood samples phagocytophilum and in the acute phase of infection may from cats were submitted for PCR testing at the commer- form morula inclusions in neutrophils identifiable on a cial laboratory. A phagocytophilium DNA was amplified blood smear.11,19 There are 20 reported cases identifying from the blood of 40 (0.92%) samples. Historical and morulae in naturally infected cats in Europe: 15 cats in clinical data were available for 16 cats. Italy,19 two cats in Poland21 and one cat each in Sweden,11 Of the 16 cats, six were spayed females, nine were Switzerland17 and Finland.18 To date, only five naturally neutered males and one was an intact male. The median infected cats with A phagocytophilum have been described age of the cats was 2 years (mean 4.1 years; range in North America.20 4 months to 13 years). All cats had access to the outdoors The objectives of this study were to collect retrospec- and resided in areas considered endemic for Ixodes spe- tively and describe the clinical and historical findings in cies. Of the 16 cats, four had been prescribed fipronil cats that were positive by PCR for A phagocytophilum (Frontline and Frontline Plus; Merial) and two had been DNA in their blood, and to describe treatment and prescribed selamectin (Revolution; Zoetis). response. We were also able to characterize intracellular The cats resided in Connecticut (n = 7), New Jersey morulae identified in neutrophils on microscopic exami- (n = 3), New York (n = 3), Massachusetts (n = 2) and nation of peripheral blood smears. Vermont (n = 1). The month of diagnosis was June (five cats), May and October (three cats each) and one cat Materials and methods each in April, July, August, September and November. Cats that had A phagocytophilum DNA amplified from a Duration of clinical signs prior to presentation to a vet- blood sample assayed at a commercial diagnostic labora- erinarian was reported for five cats (median 2 days; tory (Antech Diagnostics, Lake Success, NY, USA) mean 2.8 days; range 1–7 days). Four cats were presented between May 2009 and May 2011 were identified by an more than once to a veterinarian prior to diagnosis of A electronic record search. The laboratory uses EDTA- phagocytophilum infection and initiation of specific treat- anticoagulated blood samples to attempt to amplify ment. Lethargy (16 cats) and elevated body temperature DNA of A phagocytophilum, henselae, Bartonella (15 cats) were the most common abnormalities reported clarridgeiae, Bartonella quintana, Ehrlichia species, the day the blood sample was collected for performance Mycoplasma haemofelis, ‘Candidatus Mycoplasma haemo- of the PCR assay (Table 1). The body temperatures for minutum’, ‘Candidatus Mycoplasma turicensis’, Rickettsia the 15 febrile cats ranged from 39.6–41.5ºC, with a mean rickettsii and using a proprietary PCR assay of 40.3°C. (FastPanel) with the standard operating procedures of Of the 16 cats, only one was concurrently positive for the laboratory. The main Genbank sequence identifica- DNA of other agents (M haemominutum and B clar- tion used for the proprietary primer design for A phago- ridgeiae). Results of a complete blood count and serum cytophilum was gb CP006618.1. The sensitivity of the biochemical panel were available for 11 cats, performed multiplex primer systems was analyzed using titered, on or immediately before the day the PCR assay was synthesized DNA sequences in bacterial plasmids. These submitted. Abnormalities are listed in Table 2. These 11

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Table 1 Clinical abnormalities reported for 16 cats the day blood was submitted and proven to contain Anaplasma phagocytophilum DNA

Clinical abnormalities Number of cats (%)

Lethargy 16 (100) Fever 15 (94) Anorexia 14 (88) Ocular signs (bilateral ocular 7 (44) discharge, elevated nictitating membranes, mild conjunctivitis) Abdominal discomfort 4 (25) Tachycardia 3 (19) Hepatosplenomegaly 1 (6) Ataxia 1(6) Figure 1 Peripheral blood smear of a cat infected with Anaplasma phagocytophilum, stained with Wright-Giemsa stain. Black arrow shows intracellular morula in the cytoplasm of a neutrophil

Table 2 Clinicopathologic abnormalities in cats infected with Anaplasma phagocytophilum

Parameter n Median* Value or range Reference interval

Total WBC (×109/l) 1 NA 2.3 3.5–16.0 RBC (×1012/l) 2 NA 5.88–10.23 5.92–9.93 Hematocrit (l/l) 2 NA 0.186–0.245 (18.6–24.5%) 0.29–0.48 (29–48) Platelets (×109/l )† 7 120 68–187 200–500 Neutrophils, PMNs (×109/l) 2 NA 1600–2430 2500–8500 Lymphocytes (×109/l) 6 498 86–936 1200–8000 Glucose (mmol/l) 2 NA 10.3–15.6 (186–281 mg/dl) 3.5–9.4 (64–170) Sodium (mEq/l) 1 NA 144 145–158 Cholesterol (mmol/l) 1 NA 10.09 (390 mg/dl) 1.94–5.69 (75–220) Blood urea nitrogen (mmol/l) 1 NA 9.28 (13 mg/dl) 10.0–25.7 (14–36) Albumin (g/l) 1 NA 40 (4.0 g/dl) 25–39 (2.5–3.9)

*A median was not able to be generated when fewer than three animals had the abnormality †Platelet clumping precluded an accurate determination of a platelet count in all seven cats. Five of the seven cats were estimated to have adequate platelets WBC = white blood cells; RBC = red blood cells; PMN = polymorphonuclear neutrophil; NA = not available cases had blood smears reviewed by medical technicians Treatment information was available for 15 cats. An and some were additionally reviewed by a board-certi- antibiotic other than doxycycline (enrofloxacin, clinda- fied veterinary clinical pathologist (PE). In three cases, mycin, amoxicillin with clavulanic acid or ampicillin) cytoplasmic inclusions were identified in 5–20% of neu- was administered to eight cats prior to amplification of A trophils. The inclusions were basophilic and intracyto- phagocytophilum DNA from blood; for two cats more than plasmic with a coarsely granular texture ranging in size one antibiotic was used. Once PCR assay results con- from 1–2 µm (Figure 1), consistent with morulae of firming the presence of A phagocytophilum DNA were Ehrlichia species or Anaplasma species. As the inclusions available, all 15 cats were administered doxycycline were not seen in other leukocytes, it was concluded they orally. Dosing information was available in four cats were most likely those of A phagocytophilum, which was (5 mg/kg PO, once or twice daily). Clinical abnormali- confirmed by PCR assay. The three cats with morula ties resolved after initiating doxycycline therapy in the identified were all hyperthermic. One had a body tem- 14 cats with a known response to treatment. The dura- perature above the mean (41°C), while the other two had tion of doxycycline administration varied from 21–45 lower body temperatures at 39.8°C and 40.2°C at the days, with most cats treated for 21 days. A phagocytophi- time of examination. Urinalyses were performed in four lum serologic test results are unknown and results of cases and proteinuria was identified by dipstick in two repeated laboratory testing and PCR assay are not avail- cases; quantitative results are not available. able. Supportive care was prescribed to seven cats,

Downloaded from jfm.sagepub.com at ANGELL ANIMAL MEDICAL CTRS on January 1, 2016 4 Journal of Feline Medicine and Surgery including subcutaneous or intravenous crystalloid fluids species.27 The seasonal distribution of feline infections (seven cats), histamine-2 receptor blocker (famotidine; mimics that seen in other species, with the greatest inci- two cats), appetite stimulant (mirtazipine; one cat) and/ dence in the late spring and early autumn. This coincides or analgesics (buprenorphine and oxymorphone; two with the greatest nymphal and adult tick activity.4,28,29 cats). The primary clinical abnormalities of lethargy, ele- vated body temperature and anorexia are consistent Discussion with those found commonly in other infected cats, dogs Cats in endemic areas are commonly exposed to A phago- and humans.8,11,15 Whether the other reported abnormal- cytophilum based on antibody prevalence rates.22 For ities presented in Table 1 are related to A phagocytophilum example, in Connecticut, antibodies against A phagocyt- infection is unknown and could not be ascertained in ophilum were detected in 38% of 93 feline sera tested by this retrospective study. The ocular changes reported enzyme-linked immunosorbent assay (ELISA).22 A sur- were non-specific and likely the result of systemic vey of different areas of the USA showed a prevalence inflammation.30 Abdominal discomfort was reported in ranging from 0–13.0%, with an overall prevalence of 4/16 cats but the cause was not determined. Radiographic 4.3%.23 Lastly, antibody prevalence rates in healthy cats hepatosplenomegaly was found in one cat in this study and cats with clinical signs (fever, lethargy, inappetance) but as hepatic and splenic aspirates were not performed living in Maine were 3.6% and 15.4%, respectively.24 The the cause is not known. study described here is the largest A phagocytophilum With the exception of identifying intracytoplasmic molecular study from feline blood samples reported to morula within neutrophils, the clinicopathologic abnor- date. In contrast to the above-reported prevalence malities are non-specific for infection with A phagocyt- rates,22–24 the prevalence rate of 0.92% in this study ophilum. In this study, anemia was identified in two cats. (40/4339 cats) is lower. The prevalence reported here The cat with the lowest hematocrit (0.186 l/l, 18.6%) was likely underestimates the true prevalence rate in specific a 4-month-old kitten. As kittens normally have a lower regions and populations of cats. The samples were sub- hematocrit than adult cats, the anemia in this case was mitted for a commercial PCR assay and included sam- not as severe as would be expected if present in an adult ples from all regions of the USA, not exclusively Ixodes cat. Anemia has only been described in one other report species endemic areas. Some veterinarians may have of cats infected with A phagocytophilum.21 Lymphopenia submitted the PCR assay for reasons not specific to A has been previously identified,11,18 and in this report was phagocytophilum. The test may have been performed to seen in six (55%) cats. Although thrombocytopenia was screen healthy cats as blood donors or to test cats with reported in 7/11 cases, all seven samples had clumped clinical syndromes not likely to be induced by A phagocy- platelets, which falsely decreases the automated platelet tophilum. All 16 of the cats identified in this study were counts.31 The absence of confirmed thrombocytopenia in positive for A phagocytophilum DNA in blood, were from this study is inconsistent with previous reports where an endemic area, had potential exposure to Ixodes spe- 3/5 cats were reportedly thrombocytopenic; however, cies, and the majority had clinical and laboratory evi- one of the three samples had clumped platelets.20 A more dence of anaplasmosis, as well as apparent responses to recent report of A phagocytophilum infection in three cats doxycycline. Only one cat had molecular evidence of in Poland described thrombocytopenia in all three, other infectious agents that may have responded to tet- though no evaluation of platelet clumping was per- racyclines. Thus, we believe that anaplasmosis was the formed.21 Though thrombocytopenia is a commonly likely diagnosis for these cats. However, the major limi- reported finding of A phagocytophilum infection in tations to the study include the variability in the diag- dogs,14,16 it may not be as common in cats. Automated nostic workup, the lack of clinicopathologic data from all platelet counts can falsely lower platelet counts owing to cats and differences in clinical management amongst the tendency of feline platelets to aggregate in vitro;31 cases. As the study was retrospective, information therefore, more studies are needed to determine if a true regarding A phagocytophilum titers or resolution of labo- thrombocytopenia exists and how frequently this abnor- ratory abnormalities after treatment could not be mality occurs. Mild hyperglycemia was identified in two determined. cats and attributed to a normal physiologic stress To date, cats with suspected anaplasmosis in the USA response. Other mild and non-specific serum biochemi- have only been described in the Northeast. In contrast, cal changes were only seen in one cat each, the conse- granulocytic anaplasmosis in dogs and humans have quences of which cannot be elucidated based on these been reported in the Midwest, West and Northeast.4,25,26 few abnormalities. Causes of this inconsistency in geographic distribution Morulae were identified in neutrophils in 3/11 (27%) between cats and dogs/humans may include insufficient cases in this report, with inclusions in approximately data, differences in the genetic strains that infect these 4–20% of neutrophils. Identification of these inclusions species and the range of the organism that affects different prompted submission of blood for the commercial PCR

Downloaded from jfm.sagepub.com at ANGELL ANIMAL MEDICAL CTRS on January 1, 2016 Savidge et al 5 test, confirming the diagnosis of A phagocytophilum infec- treatment prior to the availability of results. When tion. Morulae appear as basophilic intracytoplasmic response to treatment with doxycycline is recorded, all inclusions that must be carefully differentiated from responded without recurrence of clinical signs. Döhle bodies. Morulae have been described repeatedly Information regarding persistent or recurrent infec- in dogs,32 horses,7 sheep33 and other ruminants,34 tions in cats is not available from this study. Persistent humans,28 European cats11,17–19 and specific pathogen- infections have been described in dogs,37 horses38 and free cats infested with wild-caught I scapularis.9 But, to sheep39 where experimentally induced clinical disease our knowledge, this is the first report of intracellular was acute and self-resolving with persistent yet cyclical morulae being seen in the neutrophils in naturally non-clinical bacteremia. Studies are needed to determine infected North American cats. The identification of mor- if feline anaplasmosis is a self-resolving disease and to ulae demonstrates the importance of manual examina- determine if clinical persistent infections occur. tion of a blood smear from any patient where granulocytic In this report all the cats were in Ixodes species anaplasmosis is considered a differential diagnosis. endemic areas with outdoor access. Routine use of tick Co-exposure with Bartonella burgdorferi and A phago- preventatives in dogs can reduce the incidence of A cytophilum occurs in dogs, horses, cats, humans and phagocytophilum transmission.40 Six cases in this report wildlife as both agents have the same vector and can had a history of parasiticide application; however, the share mammalian hosts.35 Different prevalences of co- last date of product application is unknown. Based on exposure in cats have been reported in different geo- this information, cats living in an Ixodes species area with graphical areas. In one study in Connecticut, 16% of cats exposure to Ixodes species might benefit from monthly had evidence of co-exposure to B burgdorferi and A phago- treatment with an effective and safe parasiticide. cytophilum,22 whereas in a study in Maine, 5.1% of ill cats It is unknown if recurrent infections occur in cats and and 2.4% of healthy cats had exposure to both infectious the most appropriate duration of doxycycline treatment agents.24 The clinical impact of co-exposure or co-infec- in cats for treating anaplasmosis is still to be elucidated. tion with B burgdorferi and A phagocytophilum is unknown Other reports have shown feline exposure to A phagocyt- in cats. In this study, as specific testing was not per- ophilum without known clinical disease;22,24 additional formed, we could not determine whether B burgdorferi studies are needed to identify how common subclinical infection contributed to the clinical disease in the cats infections occur. To date, all cats in the USA with clinical described. disease have been reported in the Northeast, despite the As previously discussed, only one cat in this study fact that A phagocytophilum is also present in the Midwest had proven (B clarridgeiae and ‘Candidatus and West. Studies evaluating feline infection of different M haemominutum’). This cat was not anemic, and as strains or geographical variants of A phagocytophilum are hemoplasmas do not always cause clinical illness,36 the needed to determine if one strain is more successful at effects of these co-infections is unknown. infecting cats than others and which strains cause clini- As a retrospective study, there was variability in the cal disease. treatment of each case. Fifteen of the 16 cats were treated with doxycycline once an infection with A phagocytophilum Conclusions was identified. Where a dose was recorded, cats received A phagocytophilum infection should be included on a dif- 5 mg/kg q12h. The duration of treatment with doxycy- ferential diagnoses list for any cat that lives in an Ixodes cline was variable (21–45 days) and clinician dependent. species endemic area with potential tick exposure and As a retrospective study it is a limitation that an explana- presents with acute or intermittent, vague symptoms of tion for the duration of treatment is unknown. The lethargy, fever and anorexia. A phagocytophilum infection majority of cats were treated for 21 days; those that were can be identified by documenting DNA in peripheral treated for 45 days were all seen at the same practice. blood by PCR prior to antibiotic administration or by Previous reports in cats have recommended treatment identifying morulae within neutrophils on a direct blood 20 with 5 mg/kg doxycycline PO q12h for 28 days. The smear examination. Exposure can be documented by ideal duration of treatment with doxycycline in cats is demonstrating the presence of antibodies with ELISA unknown and warrants additional study. One cat and indirect fluorescent antibody serology; a fourfold received enrofloxacin prior to testing. The cat initially change in convalescent titers after 14 days indicates an improved with treatment, but had recurrence of clinical active infection.8 Treatment without signs of recurrence signs and tested positive, demonstrating the importance has been successful with tetracyclines. of identification of this infection and appropriate treat- ment. All other non-tetracycline antibiotics were admin- Acknowledgements We would like to thank Sheri Ihle istered after sample collection while awaiting test results. and Pamela Mouser for their editorial support with this Owing to the retrospective nature of this study it is paper. All work was performed at Angell Animal Medical unknown if cats responded to non-tetracycline Center, Boston MA 02130, USA.

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