RH: SIM ET AL.—CLINICAL ANAPLASMOSIS IN PRZEWALSKI’S HORSES 1 CLINICAL DISEASE ASSOCIATED WITH ANAPLASMA PHAGOCYTOPHILUM 1 INFECTION IN CAPTIVE PRZEWALSKI’S HORSES (EQUUS FERUS PRZEWALSKII) 2 3 Richard R. Sim, D.V.M.; Priscilla H. Joyner, B.V.M.S., Dipl. A.C.Z.M.; Luis R. Padilla, 4 D.V.M., Dipl. A.C.Z.M.; Paul Anikis, D.V.M.; and Copper Aitken-Palmer, D.V.M., Ph.D., Dipl. 5 A.C.Z.M. 6 7 From the Wildlife Center of Virginia, PO Box 1557, Waynesboro, Virginia 22980, USA 8 (Sim); Department of Conservation Medicine, Smithsonian Conservation Biology Institute, 1500 9 Remount Road, Front Royal, Virginia 22630, USA (Padilla, Joyner, Aitken-Palmer); and 10 Piedmont Equine Practice, 4122 Zulla Road, The Plains, Virginia 20198, USA (Anikis). Present 11 addresses (Sim): Birmingham Zoo, Inc., 2630 Cahaba Road, Birmingham, Alabama 35223, 12 USA; (Padilla): Saint Louis Zoo, 1 Government Drive, Saint Louis, Missouri 63110, USA; 13 (Aitken-Palmer): Chicago Zoological Society, Brookfield Zoo, 3300 Golf Rd, Brookfield, 14 Illinois 60513, USA. 15 16 Corresponding author: Richard R. Sim, Birmingham Zoo, Inc., 2630 Cahaba Road, 17 Birmingham, Alabama 35223, USA; sim.richardr@gmail.com.18 RH: SIM ET AL.—CLINICAL ANAPLASMOSIS IN PRZEWALSKI’S HORSES 2 Abstract: Anaplasma phagocytophilum is a tick-borne pathogen of domestic horses and the 19 causative agent of Equine Granulocytic Anaplasmosis. This case series describes three 20 confirmed cases of clinical anaplasmosis, and a fourth case of presumptive anaplasmosis in 21 Przewalski’s horses (Equus ferus przewalskii) housed at the Smithsonian Conservation Biology 22 Institute from 2008 – 2014. Clinical signs varied among individuals with affected horses 23 exhibiting lethargy, weakness, pyrexia, hypophagia, reluctance to move, or ataxia. 24 Anaplasmosis was confirmed with a combination of identification of neutrophilic inclusions 25 (morulae) on peripheral blood smear, positive polymerase chain reaction (PCR) testing of whole 26 blood, or convalescent titers. All animals recovered after antimicrobial therapy with 27 oxytetracycline. Diagnosis should be made by a combination of clinical signs plus identification 28 of morulae or positive A. phagocytophilum PCR. Disease is curative with treatment using 29 oxytetracyline intramuscularly or intravenously followed by daily therapy with oxytetracyline or 30 minocycline for 14 – 30 days. The authors recommend that A. phagocytophilum infection be 31 included on any differential list for Przewalski’s horses presenting with fever or ataxia within or 32 near an enzootic area. 33 34 Key words: Anaplasma phagocytophilum, anaplasmosis, Equus ferus przewalskii, PCR, 35 Przewalski’s horse. 36 37 RH: SIM ET AL.—CLINICAL ANAPLASMOSIS IN PRZEWALSKI’S HORSES 3 INTRODUCTION 38 39 The Przewalski's horse (P-horse; Equus ferus przewalskii) is a subspecies of the wild 40 horse (Equus ferus) that is native to central Asia. P-horses became extinct in the wild in the late 41 1960s primarily due to habitat loss, hunting, and competition with domestic livestock.6 With the 42 assistance of ex-situ conservation programs, the species has been successfully reintroduced to 43 Mongolia and China, and is now classified as endangered by the International Union for 44 Conservation of Nature.6 45 The Smithsonian Conservation Biology Institute (SCBI) in Virginia, USA has 46 contributed to P-horse ex-situ conservation for nearly four decades with P-horses housed in 47 modified domestic animal barns with access to grazing pastures surrounded by forest and 48 agricultural fields. Annual vaccination, at the time of this manuscript development, has been 49 consistent for the last decade, and includes a killed rabies virus and Neorickettsia (Ehrlichia) 50 risticii bacterin combination vaccine (POTOMAVAC + IMRAB, Merial Limited, Inc., Duluth, 51 Georgia 30096, USA), and a killed West Nile Virus (WNV), eastern equine encephalomyelitis 52 virus (EEE), western equine encephalomyelitis virus (WEE), and tetanus toxoid combination 53 vaccine (WEST NILE-INNOVATOR +EWT, Zoetis, Florham Park, New Jersey 07932, USA). 54 Anaplasma phagocytophilum (formerly Ehrlichia equi) is an emerging tick-borne, non-55 contagious pathogen that causes Equine Granulocytic Anaplasmosis (EGA, formerly Equine 56 Granulocytic Erhlichiosis) in domestic horses, and Human Granulocytic Anaplasmosis.2,8 This 57 pathogen is seasonally transmitted by the Ixodes spp. tick vector and has a worldwide 58 distribution throughout North America, Europe, Africa, and Asia.3,8,11 This gram-negative cocci 59 rickettsial organism has a tropism for granulocytes.8 Acute infection results in the formation of 60 RH: SIM ET AL.—CLINICAL ANAPLASMOSIS IN PRZEWALSKI’S HORSES 4 morulae, which are granular aggregates (inclusion bodies) within the cytoplasm of neutrophils 61 that stain bluish-gray with Giemsa or Wright-Leishman stains.3,8,11 Domestic horse EGA was 62 first described in 1969 in California, USA, and recently has been reported in Colorado, 63 Connecticut, Florida, Illinois, Minnesota, Virginia, and Wisconsin.3,7,8 Ticks of varying species 64 are present throughout the SCBI property including I. scapularis, and A. phagocytophilum is 65 endemic in this region.1 66 Clinical disease in multiple P-horses at SCBI with evidence of A. phagocytophilum 67 infection prompted a retrospective review of medical records for cases of anaplasmosis. The 68 medical records of 31 P-horses housed at SCBI from 2008 to 2014 were reviewed to develop a 69 description of clinical anaplasmosis in P-horses, including diagnostic methods and treatments. 70 Data compiled from the records included gender, age, presenting signs, physical examination 71 findings, diagnostic testing results, treatment, and outcome (Tables 1-2). From 2008-2014, three 72 confirmed cases of clinical anaplasmosis in captive P-horses were diagnosed and treated at 73 SCBI; one of these P-horses was diagnosed with A. phagocytophilum re-infection or 74 recrudescence five years after the initial diagnosis. A fourth case of presumptive anaplasmosis 75 was tentatively diagnosed based on clinical signs, exclusion of other etiologies, and response to 76 empirical therapy. 77 78 CASE REPORTS 79 80 Case 1 81 82 RH: SIM ET AL.—CLINICAL ANAPLASMOSIS IN PRZEWALSKI’S HORSES 5 An estimated 14-yr-old, female P-horse (238 kg) that had been in the collection for five 83 months presented for acute lethargy and severe ataxia in hind limbs in July 2008. The animal 84 had no known clinically relevant history. At presentation, the horse collapsed into lateral 85 recumbency while being transported to the onsite veterinary hospital for evaluation. Under 86 chemical restraint, the animal was examined revealing dehydration (tacky mucous membranes 87 and prolonged capillary refill time) and pyrexia (rectal temperature 103 ˚F). The P-horse was 88 administered crystalloid fluid therapy (5% Dextrose and 0.9% Sodium Chloride Injection; 89 Abbott Laboratories, Abbott Park, Illinois 60064, USA; 11 L i.v.), ceftiofur sodium (Naxcel; 90 Zoetis; 4.2 mg/kg i.v.), flunixin meglumine (Banamine; Merck Animal Health, Kenilworth, New 91 Jersey 07033, USA; 1.3 mg/kg i.v.), vitamin E (Natural E-300; Neogen Corp., Lexington, 92 Kentucky 40511, USA; 12.6 IU/kg s.c.), and dexamethasone sodium phosphate (Dexium-SP; 93 Bimeda Inc., Riverside, Missouri 64150, USA; 0.13 mg/kg s.c.). 94 Contusions were noted following placement of a jugular intravenous catheter and at other 95 injection and phlebotomy sites during the exam. Following anesthetic recovery, the animal was 96 unable to stand, so was supported with a sling positioned under the ventrum with its hooves 97 contacting the ground. A complete blood count (CBC) and serum chemistry panel revealed 98 leukopenia due to a lymphopenia (WBC count 4.1 x103 cells/μl; reference range 4.8 - 12.36 x103 99 cells/μl; lymphocyte count 0.5 x103 cells/μl; reference range 0.87 - 5.58 x103 cells/μl), 100 thrombocytopenia (55 x103 cells/μl; median 190 cells/μl, range 14 – 474 x103 cells/μl), and 101 hypophosphatemia (1.7 mg/dL; reference range 2.2 – 7.8 mg/dL).16 102 Morulae were identified within neutrophils by microscopic evaluation of a peripheral 103 blood smear, resulting in a diagnosis of presumptive anaplasmosis. Oxytetracycline HCl 104 (Oxytet-100; Norbrook Inc., Lenexa, Kansas 66219, USA; 8.4 mg/kg i.m. q. 12 hr for 10 days) 105 RH: SIM ET AL.—CLINICAL ANAPLASMOSIS IN PRZEWALSKI’S HORSES 6 was initiated that day. Polymerase chain reaction (PCR) assays on whole blood were positive for 106 A. phagocytophilum and negative for N. risticii (Cornell Animal Health Diagnostic Center 107 [CAHDC], Ithaca, New York 14853, USA). By serum neutralization (SN), the P-horse was 108 seropositive for WNV (1:768; CAHDC). Enzyme-linked immunosorbent assay (ELISA) tests 109 revealed the P-horse was seronegative for EEE and WEE viruses (Texas A&M Veterinary 110 Medical Diagnostic Laboratory, College Station, Texas 77843, USA). 111 Improved alertness and responsiveness were noted the day after presentation, but ataxia 112 persisted, and the P-horse remained sling-assisted due to generalized weakness. A recheck CBC 113 revealed a mild neutrophilic leukocytosis (17.6 x103 cells/μl; neutrophil count 14.6 x103 cells/μl; 114 reference range 1.74 - 8.05 x103 cells/μl) with lymphopenia (0.4 x103 cells/μl) and monocytosis 115 (2.5 x103 cells/μl; reference range 0.06 – 0.66 x103 cells/μl).16 Thrombocytopenia persisted (77 116 x103 cells/μl). The P-horse’s attitude was considered normal at day 4 as the animal was 117 unapproachable without sedation. Eight days after presentation, standing sedation was 118 implemented for follow-up venipuncture. A CBC revealed persistent thrombocytopenia (81 x103 119 cells/μl), and morulae were no longer observed in the neutrophils. The animal was PCR-120 negative for A. phagocytophilum (CAHDC). Ten days after presentation, the P-horse was no 121 longer showing any clinical signs. 122 Following this episode of clinical anaplasmosis in 2008, the P-horse remained clinically 123 healthy for several years. One year after infection, this animal was PCR-negative (CAHDC) and 124 seronegative (<1:20) for A. phagocytophilum by IFA (University Tennessee College of 125 Veterinary Medicine Diagnostic Laboratory Services [UTDLS], Knoxville, Tennessee 37996, 126 USA). The UTDLS laboratory considers titers ≥ 1:80 to be moderate to high in level of antibody 127 and likely more indicative of current or recent exposure. For the purposes of this manuscript, 128 RH: SIM ET AL.—CLINICAL ANAPLASMOSIS IN PRZEWALSKI’S HORSES 7 titers ≥ 1:80 will be referred to as positive. Three years from initial presentation, it had 129 seroconverted (1:1280; UTDLS) with no evidence of infection in the intervening years. 130 Five years after the original clinical diagnosis of anaplasmosis, this individual (19-yr-old, 131 277-kg), presented with a 24-hour history of moderate bilateral ataxia in the hind limbs and 132 conscious proprioceptive deficits. The horse was restrained in a hydraulic mechanical restraint 133 device (Fauna Hydraulic TAMER, Fauna Research Inc., Red Hook, New York 12571, USA) for 134 exam with a towel as an eye cover, and found to be quiet, alert, responsive, and euthermic. 135 Blood was collected. CBC and serum chemistry results were unremarkable with platelets scored 136 as ‘adequate,’ but not quantified. No morulae were seen on microscopic examination of the 137 blood smear. Despite laboratory findings, anaplasmosis was suspected and long-acting 138 oxytetracycline (Noromycin 300 LA; Norbrook Inc.; 10 mg/kg i.v.), flunixin meglumine (1.1 139 mg/kg i.v. once, then p.o. q. 24 hr for 7 days), and vitamin E (10.2 IU/kg s.c.) were 140 administered. Three days after presentation, therapy continued with oral minocycline HCl 141 (Ranbaxy Pharmaceuticals Inc., Jacksonville, Florida 32257, USA; 100 mg capsules; 4.1 mg/kg 142 p.o. q. 12 hr for 28 days). Ataxia was mild by eight days after presentation. The horse was 143 considered neurologically normal one month after presentation. Paired convalescent titers 144 showed a four-fold increase in A. phagocytophilum from presentation to 36 days later (1:320 145 versus 1:1280; UTDLS), and the animal was PCR-negative at both of these timepoints 146 (CAHDC). 147 148 Case 2 149 150 RH: SIM ET AL.—CLINICAL ANAPLASMOSIS IN PRZEWALSKI’S HORSES 8 A 21-yr-old, male P-horse (345 kg) presented with a 24-hour history of lethargy and 151 ataxia in October 2009. The animal had been in the SCBI collection for 1.5 years prior to 152 presentation with no relevant medical history. On presentation, the animal exhibited depression, 153 ataxia in all four limbs, and decreased blink reflexes on the right side. Under chemical restraint, 154 the P-horse was examined, revealing pyrexia (rectal temperature 103.7 ˚F). Cytology from a 155 mucus sample off the endotracheal tube revealed moderate suppurative and histiocytic 156 inflammation, and very rare, morulae in the neutrophils. Blood was collected; a CBC and serum 157 chemistry revealed marked thrombocytopenia (38 x103 cells/μl) and hypophosphatemia (1.3 158 mg/dL). Morulae were observed, but rarely, in neutrophils in a peripheral blood smear. Lateral 159 cervical vertebral radiographs were unremarkable. The P-horse was administered 160 oxytetracycline HCl (11.6 mg/kg, i.m.), flunixin meglumine (0.58 mg/kg i.v.), vitamin E (8.7 161 IU/kg s.c.), dexamethasone sodium phosphate (0.012 mg/kg i.v.), and crystalloid fluids (Lactated 162 Ringer’s Injection USP; Abbott Laboratories; 6 L i.v.). 163 Anaplasmosis was diagnosed in this P-horse by observation of morulae, positive PCR 164 testing for A. phagocytophilum (CAHDC), and negative IFA (<1:20; UTDLS). The P-horse was 165 found to be negative for equine herpesvirus-1 (EHV; SN, 1:24), EHV-2 (SN, 1:48), and N. 166 risticii (IFA, <1:200) (CAHDC). The P-horse was negative for WNV acute infection (IgM 167 capture ELISA, 2.125), and a high serum neutralization titer (1:1536) demonstrated strong 168 humoral response, likely from vaccination (CAHDC). The P-horse was seropositive for 169 Sarcocystis neurona (IFA, 1:640, CAHDC). 170 Within hours of treatment the P-horse was bright, alert, and responsive with mild ataxia 171 in all limbs. Oxytetracycline HCl administration continued for 7 days (5.8 mg/kg i.m., q. 12 hr 172 RH: SIM ET AL.—CLINICAL ANAPLASMOSIS IN PRZEWALSKI’S HORSES 9 for 2 days, then q. 24 hr for 5 days). Clinical resolution of neurological signs occurred four days 173 after presentation/initial treatment with no residual ataxia or other neurological abnormalities. 174 175 Case 3 176 177 A 24-yr-old, male P-horse (273 kg) presented with a three-day history of hypophagia, 178 lethargy, and hind limb ataxia in November 2012. The horse had been in the collection for 12 179 years and had no relevant medical history. With a dull attitude, poor appetite, and hind limb 180 ataxia, empirical therapy with minocycline HCl (4.4 mg/kg p.o. q. 12 hr for 14 days) was 181 initiated. After three days with minimal improvement the animal was chemically restrained for 182 evaluation, and there were no relevant findings on examination. Lateral cervical radiographs 183 revealed moderate osteoarthritis at C4/C5 and C5/C6, but no apparent narrowing of the spinal 184 canal. Blood was collected; a CBC and serum chemistry were unremarkable. No morulae were 185 evident on peripheral blood smear. PCR for A. phagocytophilum was negative with a moderate 186 positive titer by IFA (1:160; UTDLS). Ultrasound-guided cervical centesis was performed to 187 obtain a cerebrospinal fluid (CSF) sample. The CSF was considered normal compared to the 188 domestic horse: clear in character with very low cellularity (WBC 1.1 cells/μl, RBC 1.1 cells/μl) 189 and a specific gravity of 1.006.14 Sarcocystis neurona Surface Antigen (SAG) 2/3/4 assays 190 revealed a serum:CSF titer ratio of 200, which indicates exposure, but not active infection in the 191 domestic horse (Equine Diagnostic Solutions [EDS], Lexington, Kentucky 40511, USA), and 192 was interpreted the same in this P-horse. 193 The animal was administered oxytetracycline HCl (10 mg/kg i.v.), vitamin E (11 IU/kg 194 s.c.), and ceftiofur crystalline free acid (Excede; Zoetis; 6.6 mg/kg s.c.). Nearly immediate 195 RH: SIM ET AL.—CLINICAL ANAPLASMOSIS IN PRZEWALSKI’S HORSES 10 improvement in mentation was noted after evaluation and treatment with intravenous 196 oxytetracycline. The following day, no ataxia was noted, but hypophagia persisted. The animal 197 was clinically normal four days after intravenous treatment with oxytetracycline began (one 198 week after onset of clinical signs). A follow up A. phagocytophilum titer conducted three years 199 later was negative (<1:20; UTDLS). 200 201 Case 4 202 203 A 1.5-yr-old, female P-horse (213 kg) presented with sudden onset lethargy and an 204 abnormal gait in April 2014. The P-horse was hanging its head low with bilateral ptosis, and had 205 a wide hind limb stance and gait. Under physical restraint in a hydraulic mechanical restraint 206 device, examination found pyrexia (rectal temperature, 106.5 ˚F), tachypnea, weight loss (10-kg 207 in one week), and dehydration. Following venipuncture to sample blood, the P-horse was treated 208 with oxytetracycline HCl (10 mg/kg i.v.), flunixin meglumine (1.1 mg/kg i.v.) and crystalloid 209 fluids (Lactated Ringer’s Injection USP; 1.5 L i.v. and 1 L s.c.). Later that day, the P-horse was 210 sedated with hydraulic restraint for further therapy, and the P-horse’s temperature and respiratory 211 rate had normalized. Crystalloid fluid therapy (0.9% Sodium Chloride Injection; Abbott 212 Laboratories; 6 L i.v.) and ceftiofur crystalline free acid (2.3 mg/kg i.m.) were administered. 213 Topical permethrin (Equi-spot; Farnam Companies, Inc., Phoenix, Arizona 85013, USA; 5 mL) 214 was applied to the withers and dorsal hindquarters for tick control. The animal was 215 neurologically appropriate with a normal gait when released from the restraint device. 216 The CBC and serum chemistry from the sample taken during the morning restraint 217 revealed hypophosphatemia (1.6 mg/dL) with morulae in the neutrophils on a peripheral blood 218 RH: SIM ET AL.—CLINICAL ANAPLASMOSIS IN PRZEWALSKI’S HORSES 11 smear and buffy coat smear. PCR for A. phagocytophilum was positive (CAHDC), and the 219 animal was seronegative by IFA (<1:20; UTDLS). A western blot for S. neurona was negative, 220 and a SAG 2/3/4 ELISA titer on serum was positive at the lowest threshold (1:250), which 221 suggests exposure but not clinical disease (EDS). WNV IgM capture ELISA was positive, which 222 was likely due to vaccination which had occurred one week prior (EDS). The Lyme Equine 223 multiplex titer revealed that the horse was negative for Borrellia burgdorferi SAGs OspA, OspC, 224 and OspF (CAHDC). 225 The following day, the P-horse was clinically normal. Treatment was continued with 226 oxytetracycline HCl (10 mg/kg i.m. q. 24 hr for 4 days) without return of clinical signs. The P-227 horse was restrained in a hydraulic mechanical restraint device for blood sampling 2, 28, and 63 228 days after presentation; the horse remained PCR-positive for A. phagocytophilum at two days, 229 but negative at 28 days after presentation (CAHDC). The P-horse did not seroconvert and was 230 titer negative at 28 and 63 days post-presentation (UTDLS). 231 232 DISCUSSION 233 234 This case series documents clinical anaplasmosis in P-horses at SCBI with clinical 235 presentations that are similar to those seen with EGA in the domestic horse. Lethargy, ataxia, 236 and pyrexia were the most common observed clinical signs in the P-horses infected at SCBI. 237 The P-horses ranged from 1.5 to 24 years of age; all older P-horses (cases 1-3, aged 14-24 years) 238 exhibited moderate to severe lethargy, severe ataxia, and mild pyrexia. The younger P-horse 239 (case 4) was noted to have mild lethargy, mild ataxia, and severe pyrexia. This demarcation of 240 clinical signs with respect to age is consistent with EGA in the domestic horse, where younger 241 RH: SIM ET AL.—CLINICAL ANAPLASMOSIS IN PRZEWALSKI’S HORSES 12 animals have been found to have less severe general clinical signs, but have a more severe 242 pyrexia.3,7 243 Spontaneous petechiation, icterus, and dependent limb edema are common clinical signs 244 of EGA in the domestic horse that were not seen in these P-horses.3,7,11 Petechiation in the 245 domestic horse is likely due to a coagulopathy secondary to consumptive thrombocytopenia.3 246 One P-horse with thrombocytopenia did exhibit multiple, iatrogenic contusions, but the other 247 case with thrombocytopenia did not show overt evidence of coagulopathy. 248 Hematologic and serum chemistry findings associated with anaplamosis in P-horses 249 differed from those seen in the domestic horse with EGA. Blood cell line aberrations were 250 minimally seen, which is markedly different findings than the pancytopenia that is common in 251 the domestic horse.11 Hypophosphatemia occurred in the P-horses with anaplasmosis; however, 252 a conclusive cause for hypophosphatemia and the clinical relevance is unclear. This finding is 253 not reported in domestic horses with EGA.3,7,8,11,14 Hypophosphatemia in the domestic horse can 254 be associated with chronic renal failure, hemoglobinuria, Brassica toxicity, inadequate dietary 255 intake, and hyperparathyroidism.14 With the exception of inadequate dietary intake, there was no 256 evidence of any of these disease processes in the P-horses evaluated. Hypophagia was 257 confirmed in one case (case 3, presumptive anaplasmosis) that did not exhibit 258 hypophosphatemia. 259 Anaplasmosis was diagnosed in 3 of 4 clinically abnormal P-horses based on the 260 identification of morulae in the neutrophils, positive A. phagocytophilum PCR, or four-fold or 261 greater increase in paired convalescent IFA titers. Importantly, all individuals with morulae in 262 their neutrophils were also PCR-positive for A. phagocytophilum and were febrile at exam. The 263 humoral response to A. phagocytophilum, as measured by IFA serology, appears less indicative 264 RH: SIM ET AL.—CLINICAL ANAPLASMOSIS IN PRZEWALSKI’S HORSES 13 of clinical disease as two of the three confirmed cases did not seroconvert, so does not have 265 much value based on one time point. Serosurveillence for A. phagocytophilum in P-horses at 266 SCBI was conducted, but is outside the scope of this report and has been reported separately.13 267 Case 1 remained seronegative one year following its initial infection, but was found to 268 have a high titer two years later without clinical signs of disease, then developed clinical disease 269 again five years after the initial infection. This individual may have undergone different 270 episodes of reinfection, recrudescence, or perhaps the magnitude of titers does not correspond to 271 the progression of this disease or antibody response in the P-horse. Historically, it has been 272 asserted that domestic horses do not maintain a chronic carrier state for A. phagocytophilum,7,11 273 but molecular persistence of the pathogen was recently demonstrated in the bloodstream of 274 asymptomatic, domestic horses for up to four months after experimental infection.4 Notably, 275 immunosuppression in the five domestic horses of this study was induced with dexamethasone 276 treatment or trailer transport before PCR-positive results were achieved.4 It is unknown whether 277 persistent A. phagocytophilum infection occurs in P-horses. 278 Follow up serologic monitoring was not standardized for this case series, but case 1, 279 during its 2013 infection event, shows that a four-fold increase between paired serum samples 280 (acute versus convalescent) is possible. Case 4 was similarly monitored, but showed no 281 seroconversion at all. In naturally infected domestic horses, a peak antibody titer occurs 19 to 81 282 days, so a rising convalescent titer is a significant finding and is a definitive means of diagnosing 283 EGA.7,11,15 Further study monitoring of convalescent titers for A. phagocytophilum in P-horses is 284 needed. 285 The ancillary diagnostics for each P-horse reported in this case series were selected based 286 on clinical judgment at the time of presentation. Investigated pathogens included: N. risticii, 287 RH: SIM ET AL.—CLINICAL ANAPLASMOSIS IN PRZEWALSKI’S HORSES 14 WNV, EEE, WEE, EHV-1, EHV-2, S. neurona, and B. burgdorferi. Ancillary testing results 288 were negative for these pathogens except for S. neurona (case 2) and WNV (cases 1, 2, and 4). 289 Case 2 had strong humoral response to S. neurona (high enough to expect natural exposure and 290 disease in domestic horses), but clinical signs improved without directed treatment for this 291 pathogen. Serologic findings for WNV were complicated by humoral immunity related to 292 routine vaccination. 293 Treatment of anaplasmosis in P-horses consisted of oral minocycline, parenteral 294 oxytetracycline, anti-inflammatory medications, and supportive therapies. In domestic horses, 295 treatment with tetracycline drugs is considered critical as this class of drugs can penetrate cells 296 and act on intracellular pathogens.3,5,11 Initiation of therapy resulted in the majority of P-horses 297 showing improvement of attitude and ataxia within 12-24 hours, and normalization of pyrexia in 298 this time period as well. In the domestic horse, it is suggested that a failure to return to normal 299 rectal temperature within 24-hours of starting tetracycline therapy is evidence that anaplasmosis 300 is not responsible for the horse’s illness.7,11 Of note, the immunomodulatory effects of 301 tetracyclines can benefit the treatment of inflammatory pathologies.5 In all the cases, 302 oxytetracycline was administered parenterally at least once. Cases 1 and 2 initially received 303 injections of oxytetracycline twice a day, which is a higher treatment frequency than is 304 recommended for the domestic horse.3,9,11 Once daily therapy of oxytetracycline in case 4 305 appeared to be effective. No adverse effects associated with oxytetracycline were noted in the P-306 horses. 307 Oral minocycline was administered to two of the four cases and was well tolerated at a 308 dosage used in domestic horses.9 Doxycycline is commonly administered as enteral therapy for 309 domestic horse EGA, but minocycline was chosen in these cases based on manufacturer 310 RH: SIM ET AL.—CLINICAL ANAPLASMOSIS IN PRZEWALSKI’S HORSES 15 availability. With high oral bioavailability, minocycline penetrates the central nervous system 311 (CNS) better than doxycycline, but, as the mechanism for ataxia in affected P-horses is 312 vasculitis-associated cerebral edema, CNS penetration is not an advantage of using 313 minocycline.7,9 Although minocycline successfully treated P-horses after initial intravenous or 314 intramuscular injection of oxytetracycline, the reverse was not true. In case 3, minocycline was 315 an initial empirical therapy, but did not result in rapid resolution of clinical signs as was seen 316 with oxytetracycline therapy. For this reason, case 3 was anesthetized three days into the disease 317 course, oxytetracycline was administered, and the individual improved rapidly afterwards. The 318 authors suggest all suspected cases of anaplasmosis in the P-horse should be initially treated with 319 at least one dose of parenteral oxytetracycline before the use of an oral tetracycline drug. 320 All clinical signs of initial infection resolved within 10 days with re-infection taking 321 longer to resolve (30 days). As such, treatment for initial anaplasmosis is recommended for 14 322 days, but in the case of re-infection treatment may need to be extended. 323 Case 3 is defined as a suspect case in this series based on a negative PCR result, but the 324 authors believe that this was a false PCR-negative result. Other differential diagnoses for 325 pyrexia and ataxia were adequately ruled out as there was no evidence of cerebral disease, 326 cervical vertebral stenotic myelopathy, or thiamine deficiency, and without xanthochromia, 327 pleocytosis, or an elevated protein level within the CSF sample, bacterial meningitis, verminous 328 meningoencephalomyelitis, and viral meningitis are of low likelihood.12,14 A normal CSF 329 sample is consistent with anaplasmosis as the pathophysiology of anaplasmosis-associated ataxia 330 is thought to be related to vasculitis and cerebral edema.7 Samples for PCR for case 3 were 331 obtained after three days of minocycline therapy and resulted in a negative PCR result. Case 4 332 was found to be PCR-positive after receiving parenteral oxytetracycline therapy for two days; 333 RH: SIM ET AL.—CLINICAL ANAPLASMOSIS IN PRZEWALSKI’S HORSES 16 whereas, case 1 was PCR-negative at eight days. Based on case 3, the authors theorize that 334 empirical therapy before samples for PCR confounded the results, and recommend that samples 335 be obtained before tetracyclines are administered if a definitive diagnosis is desired. 336 In summary, A. phagocytophilum can result in clinical anaplasmosis in P-horses with 337 similar disease description to EGA of domestic horses. In the P-horse, lethargy, ataxia, and fever 338 are the most common clinical signs. Thrombocytopenia and leukopenia occurred in the P-horse 339 with anaplasmosis but were less common findings as compared to the domestic horse, and 340 anemia was not documented. A potentially unique characteristic of anaplasmosis in the P-horse 341 is hypophosphatemia, but the clinical significance is unclear. A definitive diagnosis of A. 342 phagocytophilum infection in the P-horse can be made based on the combination of clinical signs 343 (i.e. febrile), presence of morulae in neutrophils, and PCR-positive testing. A four-fold or 344 greater rise in convalescent IFA titers is associated with clinical disease of P-horse anaplasmosis, 345 but further studies are needed to understand the immunology of anaplasmosis in the P-horse. 346 Serology should be considered to have limited utility if measured at only one timepoint. Similar 347 to the domestic horse, initiation of tetracycline therapy and supportive care in P-horses results in 348 rapid improvement of clinical signs. Based on this case series, oxytetracyline (8.4 – 11.6 mg/kg 349 i.v. or i.m.) once followed by once daily injectable oxytetracycline or twice daily oral 350 minocycline (4.1 – 4.4 mg/kg) is recommended for the treatment of anaplasmosis in P horses. A. 351 phagocytophilum infection should be included as a differential for ataxia and pyrexia in P-horses 352 housed within or near an enzootic area. 353 354 Acknowledgements: The authors gratefully acknowledge the assistance of the animal care 355 staff of the Smithsonian Conservation Biology Institute and the clinical pathology staff of the 356 RH: SIM ET AL.—CLINICAL ANAPLASMOSIS IN PRZEWALSKI’S HORSES 17 Smithsonian’s National Zoological Park. Specifically, the authors would like to thank Ann 357 Bratthauer for her expertise in clinical pathology, and Dolores Reed for her knowledge and 358 dedication to the care and husbandry of P-horses. 359 360 RH: SIM ET AL.—CLINICAL ANAPLASMOSIS IN PRZEWALSKI’S HORSES 18 LITERATURE CITED 361 362 1. Centers for Disease Control and Prevention [Internet]. Annual Cases of Anaplasmosis in the 363 United States; c2016 Jan 5 [cited 2016 July 18]. Available from: 364 http://www.cdc.gov/anaplasmosis/stats/. 365 2. 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In: ISIS 403 RH: SIM ET AL.—CLINICAL ANAPLASMOSIS IN PRZEWALSKI’S HORSES 20 Physiological Reference Intervals for Captive Wildlife: A CD-ROM Resource. Eagan (MN): 404 International Species Information System; 2013. 405 Table 1. Characterization of Anaplasma phagocytophilum infection in captive Przewalski's horses (Equus ferus przewalskii) at the Smithsonian Conservation Biology Institute in Virginia, USA between 2008-2014 with ancillary diagnostics.a Case Sex Age (yr) Onset Clinical signs CBC & morphology Serum chem. Testing for A. phagocytophilum Ancillary diagnostics PCRb IFAc Initial Follow-up Initial Follow- up 1* F 14 Jul 2008 Lethargy, severe ataxia, mild pyrexia, dehydration ↓WBC, ↓platelets, morulae in neutrophils ↓P Pos Neg (8 days) NT NT Neorickettsia risticii PCR negativeb EEE IgM ELISA negatived WEE IgM ELISA negatived WNV SN, 1:768b 19 Jun 2013 Moderate ataxia NSF NSF Neg Neg (36 days) 1:320 1:1280 (36 days) NT 2 M 21 Oct 2009 Lethargy, moderate ataxia, decreased blink reflexes, mild pyrexia ↓platelets, morulae in neutrophils ↓P Pos NT <1:20 NT EHV-1 SN, 1:24; EHV-2 SN, 1:48b N. risticii IFA, <1:200b WNV: SN, 1:1536; IgM Capture ELISA, 2.125b Sarcocystis neurona IFA, 1:640b 3 M 24 Nov 2012 Lethargy, hypophagia, ataxia NSF NSF Neg NT 1:160 NT CSF, clear, WBC 1.1 cells/μl, RBC 1.1 cells/μl & specific gravity 1.006 S. neurona SAG 2/3/4 titers: Serum, 1:1000; CSF, 1:5; Serum:CSF titer ratio, 200e 4 F 1.5 Apr 2014 Lethargy, ptosis, wide- based stance, marked pyrexia Morulae in neutrophils ↓P Pos Pos at 2 days, Neg at 28 days <1:20 <1:20 (28 & 63 days) S. neurona Western blot negative; SAG 2/3/4 ELISA titer on serum 1:250e WNV IgM capture ELISA positivee Lyme Equine multiplex titer, negative for Borrellia burgdorferi SAGs OspA, OspC, and OspFb * Individual had a recurrence of anaplasmosis five years after initial diagnosis. a CBC, Complete blood count; chem., chemistry; CSF, cerebrospinal fluid; ELISA, enzyme-linked immunosorbent assay; EEE, eastern equine encephalomyelitis; EHV, Equine herpesvirus; F, female; IgM, immunoglobulin M; IFA, indirect fluorescent antibody assay; M, male; Neg, negative; NSF, no significant findings; NT, not tested; ↓P, hypophosphatemia; PCR, polymerase chain reaction; ↓platelets, thrombocytopenia; Pos, positive; SAG, surface antigen; SN, serum neutralization assay; ↓WBC, leucopenia; WEE, western equine encephalomyelitis; WNV, West Nile Virus. b Cornell Animal Health Diagnostic Center, Ithaca, New York 14853, USA. c University Tennessee College of Veterinary Medicine Diagnostic Laboratory Services, Knoxville, Tennessee 37996, USA. d Texas A&M Veterinary Medical Diagnostic Laboratory, College Station, Texas 77843, USA. e Equine Diagnostic Solutions, Lexington, Kentucky 40511, USA. Table 2. Treatment of Anaplasma phagocytophilum infection in captive Przewalski's horses (Equus ferus przewalskii) at the Smithsonian Conservation Biology Institute in Virginia, USA between 2008-2014.a Case # Onset Antimicrobial therapy Supportive therapies Disease duration (days) 1* Jul 2008 Oxytet HCl 8.4 mg/kg i.m. q. 12 hr for 10 d Ceftiofur sodium 4.2 mg/kg i.v. BW sling support for 3 d 5% dextrose 11 L i.v. Flunixin meglumine 1.3 mg/kg i.v. Vitamin E 12.6 IU/kg s.c. Dex-SP 0.13 mg/kg s.c. 10 Jun 2013 Oxytet-LA 10 mg/kg i.v. once Minocycline HCl 4.1 mg/kg p.o. q. 12 hr for 28d, started on day 3 Flunixin meglumine 1.1 mg/kg i.v. once, then p.o. for 7 d Vitamin E 10.2 IU/kg s.c. 30 2 Oct 2009 Oxytet HCl 11.6 mg/kg i.m. once, then 5.8 mg/kg i.m. q. 12 hr for 2 d, then 5.8 mg/kg q. 24 hr for 5 d LRS 6 L i.v. Flunixin meglumine 0.58 mg/kg i.v. Vitamin E 8.7 IU/kg s.c. Dex-SP 0.012 mg/kg i.v. 7 3 Nov 2012 Minocycline 4.4 mg/kg p.o. q. 12 hr for 14 d Oxytet HCl 10 mg/kg i.v. at day 3 CCFA 6.6 mg/kg s.c. at day 3 Vitamin E 11 IU/kg s.c. 4 4 Apr 2014 Oxytet HCl 10 mg/kg i.v. once, then 10 mg/kg i.m. q. 24 hr for 4 d CCFA 2.3 mg/kg i.m. Flunixin meglumine 1.1 mg/kg i.v. LRS 1.5 L i.v. and 1 L s.c. 0.9% NaCl 6 L i.v. Permethrin 2.25 g topically 2 * Individual had a recurrence of anaplasmosis five years after initial diagnosis. a Oxytet, oxytetracycline; HCl, hydrochloride; BW, body weight; Dex-SP, dexamethasone sodium phosphate; Oxytet-LA; long-acting oxytetracycline; LRS, Lactated Ringer’s solution; CCFA, ceftiofur crystalline free acid.