ACUTE GRANULOCYTIC ANAPLASMOSIS IN A CAPTIVE TIMBER WOLF (CANIS LUPUS OCCIDENTALIS)

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1 ACUTE GRANULOCYTIC ANAPLASMOSIS IN A CAPTIVE TIMBER WOLF (CANIS LUPUS OCCIDENTALIS) Author(s): Michael Leschnik, D.V.M., Georges Kirtz, D.V.M., Zsófia Virányi, Ph.D., Walpurga Wille-Piazzai, and Georg Duscher, D.Sc. Source: Journal of Zoo and Wildlife Medicine, 43(3): Published By: American Association of Zoo Veterinarians DOI: URL: BioOne ( is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne s Terms of Use, available at terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder. BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.

2 Journal of Zoo and Wildlife Medicine 43(3): , 2012 Copyright 2012 by American Association of Zoo Veterinarians ACUTE GRANULOCYTIC ANAPLASMOSIS IN A CAPTIVE TIMBER WOLF (CANIS LUPUS OCCIDENTALIS) Michael Leschnik, D.V.M., Georges Kirtz, D.V.M., Zsófia Virányi, Ph.D., Walpurga Wille-Piazzai, and Georg Duscher D.Sc. Abstract: A 2-yr-old male captive timber wolf, kept in an outdoor enclosure in Austria, presented with anorexia, depression, and fever in June. Tick infestation was reported despite monthly acaricidal treatment. The microscopic examination of a blood smear revealed elementary bodies and morulae suspicious for Anaplasma phagocytophilum. Polymerase chain reaction confirmed the infection by the G-variant of the pathogen, also found in horses and humans but not in wild ruminants. The wolf seroconverted within 2 wk, and antibodies persisted thereafter. Therapy was started with doxycycline for 10 days, and the wolf recovered within 24 hr. Clinical symptoms and temporary changes in blood parameters (thrombocytopenia, lymphopenia, mild anemia) in this wolf show high similarity to granulocytic anaplasmosis seen in dogs. This is the first report on granulocytic anaplasmosis in a wolf, indicating that A. phagocytophilum might cause clinical disease in the wolf. Key words: Anaplasma phagocytophilum, Canis lupus occidentalis, granulocytic anaplasmosis, timber wolf. BRIEF COMMUNICATION Anaplasma phagocytophilum is a gram-negative obligatory intracellular pleomorphic bacterium with worldwide geographic distribution belonging to the order Rickettsiales. In Europe, the organism is mainly transmitted by the hard tick Ixodes ricinus to its mammalian hosts, where it infects the neutrophils. 10 Granulocytic anaplasmosis is reported in many species, like domestic ruminants, cats, dogs, and horses, as well as humans. 12 Other domestic and wildlife mammals have been found to harbor the pathogen, although clinical disease has not been described. 10 Symptomatic granulocytic anaplasmosis has been described in Austria in cats and dogs, with a canine seroprevalence of 56.5%. 4 6 A 2-yr-old male, 38-kg, captive timber wolf, kept in a pack of five wolves in an outdoor enclosure (8,000 m 2 ) at the Wolf Science Center (Wildpark Ernstbrunn, Lower Austria; latitude N, longitude E), was reported to be weak, inactive, and depressed for 2 days with a slow and uncoordinated gait. The wolf had been hand raised and received regular sessions of training and behavioral research, but on these days, it did not participate in the daily activities as From the Veterinary University Vienna, Department for Small Animals, Clinic for Internal Medicine and Infectious Diseases, 1210 Vienna, Austria (Leschnik); the Department of Pathobiology, Institute of Parasitology, Veterinary University Vienna, 1210 Vienna, Austria (Wille-Piazzai, Duscher); In Vitro Laboratory, 1030 Vienna, Austria (Kirtz); and the Wolf Science Center, 2115 Ernstbrunn, Austria (Vira nyi). Correspondence should be directed to Dr. Leschnik (michael.leschnik@vetmeduni.ac.at). usual. On the second day, the wolf refused food, and its body temperature was 418C. Tick infestation was observed from April to June, although a spot-on acaricide (permethrin imidacloprid, Advantixt, Bayer Animal Health GmbH, Leverkusen, Germany) had been applied monthly. Blood was sampled, and cell counts and a blood smear examination (eosin methylene blue) were performed (Table 1). The microscopic examination of the stained blood smear (concentration line technique) revealed multiple coccoid elementary bodies and morulae in the cytoplasm of circulating neutrophils suggestive of acute granulocytic anaplasmosis (Fig. 1). DNA extraction was performed using High Pure PCR Template Preparation Kitt (Roche Diagnostics, Mannheim, Germany) from the white blood cell pellet, which was obtained by centrifugation of 2 ml of whole ethylenediaminetetraacetic acid blood and lysis of the erythrocytes with Erythrocyte Lysis Buffert (QIAGEN GmbH, Hilden, Germany). To test for A. phagocytophilum, a 16S rrna polymerase chain reaction (PCR), which yields a 619-bp fragment, was conducted with a modified protocol. 2 The assay was reduced to 25 ll instead of 50 ll. Primers were used in a concentration of 12.5 pmol/ll each. Furthermore, 1.5 mm MgCl 2 (Promega, Mannheim, Germany), 200 lm of each deoxyribonucleotide triphosphate (dntp), U GoTagt polymerase (Promega), and 5 ll of template DNA were mixed, and PCR was carried out for 5 min at 948C, 30 cycles of 15 sec at 968C, 45 sec at 66.88C, and 60 sec at 728C, followed by 7 min elongation at 728C. Furthermore, a PCR targeting a 126-bp-long groel gene 645

3 646 JOURNAL OF ZOO AND WILDLIFE MEDICINE Table 1. values. 11 Hematological parameters of a timber wolf with acute granulocytic anaplasmosis and reference Blood parameter a Acute sample First convalescent sample Reference values PCV (L/L) RBC (10 12 /L) WBC (10 9 /L) Lymphocytes (10 9 /L) Neutrophils (10 9 /L) Monocytes (10 9 /L) Eosinophils (10 9 /L) Thrombocytes (10 9 /L) a PCV, packed cell volume; RBC, red blood cells; WBC, white blood cells. within the conserved heat shock operon groesl was modified for use with conventional PCR: In 25 ll of five times Green GoTaq reaction buffer, 1.5 mm MgCl 2 (Promega), 200 lm of each dntp, U GoTag polymerase (Promega), 25 pmol/ ll forward primer groe F2, 12.5 pmol/ll groe R1, and 1 ll of the extracted DNA described above were mixed. 8 Amplification was performed at 958C for 10 min, followed by 40 cycles 15 sec at 958C, 1 min at 608C, and 30 sec at 728C. Elongation took place at 728C for 3 min. Both DNA products were separated on 2% agarose gel after amplifying on a Biometra T gradient thermocycler. After staining with ethidium bromide, Figure 1. Multiple elementary bodies and morulae (A. phagocytophilum) in the cytoplasm of circulating neutrophils in the wolf s blood smear (concentration line technique; eosin methylene blue, Hemacolort, Merck, 1140 Vienna, Austria), 3100 oil magnification.

4 LESCHNIK ET AL. GRANULOCYTIC ANAPLASMOSIS IN A TIMBER WOLF 647 visualization was carried out under ultraviolet (UV) light. The groel sequence was analyzed by cycle sequencing capillary electrophoresis at the sequencing service (Microsynth AG, 9436 Balgach, Switzerland). The 16S rrna revealed a positive result. The analysis of the groel delivered 100% coverage of the G-variant previously described. 8 Therapy was started with doxycycline (Vibramycint, Pfizer GmbH, Karlsruhe, Germany) orally at a dosage of 5 mg/kg twice a day for 10 days. Within 24 hr, the wolf recovered completely, body temperature was within normal limits, and food uptake started again. Convalescent blood values were performed 2 wk thereafter (Table 1). No intracytoplasmic inclusion bodies were observed in the blood smear, and PCR for A. phagocytophilum was negative. Serum samples from the acute phase and from the convalescent phase (2 and 8 wk later) were examined for immunoglobulin G antibodies to A. phagocytophilum by an indirect immunofluorescence assay validated for dogs (MegaScreen FLUOANAPLASMAt; Megacore, 6912 Hoerbranz, Austria). The serological investigation revealed a negative titer (1:40) in the acute sample and a titer of 1:160 (cutoff 1:80) in both convalescent samples. Granulocytic anaplasmosis is a common tickborne infection in domestic animals and humans. To the authors knowledge, this is the first case report of a wolf with acute anaplasmosis showing high similarity to granulocytic anaplasmosis in dogs. The most common symptoms in dogs include lethargy and fever, also reported in this affected wolf. Slow and uncoordinated gait may be caused by an additional clinical manifestation, including polyarthritis or meningitis, also described in dogs. 1 Hematological changes including thrombocytopenia, lymphopenia, and mild anemia are found in dogs, evident also in the current wolf when compared with the reference values of the gray wolf. 4,11 Mild anemia persisted for at least 2 wk after diagnosis. Increasing hematocrit was not evident in the convalescent sample, perhaps because time was too short for the anemia to resolve. Seroconversion supports the confirmed diagnosis of acute anaplasmosis in the wolf, although the simple evidence of specific antibodies does not prove the causality of infection and symptomatic disease because seroprevalence in asymptomatic dogs is high. 1,4 Low antibody titers in this wolf could be due to abrogated infection by prompt treatment. Specific antibodies are reported to persist for several months. 1 The peaks in nymphal and adult tick activities over the year is reflected in the temporal occurrence of canine anaplasmosis and therefore can be taken as a risk factor. Infection in the current wolf occurred in June, with a high activity of I. ricinus. Tick infestation in the wolves has been proven, even though the spot-on treatment has been applied. Outdoor activity is also a risk factor, which in terms of this wolf is a given because the pack is kept in an outdoor enclosure with bushes and undergrowth vegetation. 1 Reducing this undergrowth vegetation in the wolves enclosure might be an additional procedure to limit future infestations. Anaplasma phagocytophilum could be divided into two lineages, showing a single nucleotide polymorphism in the groel gene. 8 Variant A (showing the base adenine at the 500-bp position) was found in roe deer, whereas the G-variant (guanine at this position) was demonstrated in horses and humans. 8 In the vector tick I. ricinus, both variants can be found, with a much higher proportion of A- variants. 8 Yet the difference of these two lineages with regard to pathogenicity or infectivity remains elusive, especially because this polymorphism is a silent mutation and the functional differences might be located elsewhere in the genome. 3 Further investigations in terms of the segregation of A. phagocytophilum variants or lineages are necessary to elucidate the natural reservoirs and to trace the infection paths of this pathogen. 7,9 Granulocytic anaplasmosis is known to be a common infectious disease in dogs, although not all animals develop clinical signs. In wildlife, symptomatic anaplasmosis also might be a rare disease. In the case of this wolf, antibiotic therapy was started immediately after diagnosis, and prompt recovery within 24 hr is more indicative of a good response to specific therapy than for spontaneous recovery by specific immune response. This case report confirms the exposure and symptomatic infection by A. phagocytophilum in a captive wolf. LITERATURE CITED 1. Carrade, D. D., J. E. Foley, D. L. Borjesson, and J. E. Sykes Canine granulocytic anaplasmosis: a review. J. Vet. Intern. Med. 23: Engvall, E. O., B. Pettersson, M. Persson, K. Artursson, and K. E. Johansson A 16S rrnabased PCR assay for detection and identification of granulocytic Ehrlichia species in dogs, horses, and cattle. J. Clin. Microbiol. 34: Haschke-Becher, E., R. Bernauer, A. M. Walleczek, P. Apfalter, S. Afazel-Saeedi, J. Kraus, G. Ladurn-

5 648 JOURNAL OF ZOO AND WILDLIFE MEDICINE er, and P. Strasser First detection of the Anaplasma phagocytophilum groel-a genotype in man. J. Infect. 60: Kirtz, G., B. Czettel, D. Thum, and E. Leidinger Anaplasma phagocytophilum in dogs in Austria: a serological prevalence study ( ). Kleintierpraxis 52: Kirtz, G., M. Meli, E. Leidinger, P. Ludwig, D. Thum, B. Czettel, S. Kölbl, and H. Lutz Anaplasma phagocytophilum infection in a dog: identifying the causative agent using PCR. J. Small Anim. Pract. 46: Kirtz, G., M. L. Meli, H. Lutz, V. Buchele, P. Niederl, P. Ludwig, B. Czettel, D. Thum, and E. Leidinger Anaplasma phagocytophilum infection in two cats in Austria. Kleintierpraxis 50: Petrovec, M., W. Sixl, R. Schweiger, S. Mikulasek, L. Elke, G. Wüst, E. Marth, K. Strasek, D. Stünzner, and T. Avsic-Županc Infections of wild animals with Anaplasma phagocytophilum in Austria and the Czech Republic. Ann. N.Y. Acad. Sci. 990: Polin, H., P. Hufnagl, R. Haunschmid, F. Gruber, and G. Ladurner Molecular evidence of Anaplasma phagocytophilum in Ixodes ricinus ticks and wild animals in Austria. J. Clin. Microbiol. 42: Silaghi, C., D. Hamel, C. Thiel, K. Pfister, L. M. F. Passos, and S. Rehbein Genetic variants of Anaplasma phagocytophilum in wild caprine and cervid ungulates from the Alps in Tyrol, Austria. Vector Borne Zoonot. Dis. 11: Stuen, S Anaplasma phagocytophilum the most widespread tick-borne infection in animals in Europe. Vet. Res. Commun. 31(Suppl. 1): Thoresen, S. I., J. M. Arnemo, and O. Liberg Hematology and serum clinical chemistry reference intervals for free-ranging Scandinavian gray wolves (Canis lupus). Vet. Clin. Pathol. 38: Woldehiwet, Z The natural history of Anaplasma phagocytophilum. Vet. Parasitol. 167: Received for publication 16 October 2011

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