In-clinic diagnosis of canine anaplasmosis based on haematological abnormalities and evaluation of a stained blood smear

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1 Original Article 1 In-clinic diagnosis of canine anaplasmosis based on haematological abnormalities and evaluation of a stained blood smear G. Kirtz; E. Leidinger INVITRO, Laboratory for veterinary diagnostics and hygiene, Vienna, Austria Keywords Dog, A. phagocytophilum, haematological abnormalities, Austria Summary Objective: Haematological changes, evaluation of a blood smear and seasonal variations may assist in an in-clinic diagnosis of acute anaplasmosis. Material and methods: Blood samples from 57 dogs were evaluated. The diagnosis of canine anaplasmosis was confirmed by detection of the microorganism by microscopic examination of a stained blood smear. Results: Thrombocytopenia was present in 5 dogs (9.%). Red blood cell count, packed cell volume and haemo - globin values were below the reference interval in 3.%,.5% and.3% of affected dogs respectively. Based on reticulocyte counts, a non-regenerative anaemia was present in 7.% of A. phagocytophi - lum-positive dogs. Leukopenia, lymphopenia, neutropenia, monocytosis and eosinophilia were present in 35.1%,.7%,.9%, 5.7% and 15.% of the dogs respectively. Conclusion: The results demonstrate that together with clinical signs, a tentative diagnosis of canine anaplasmosis can be made based on typical haematological changes. The most common haematological abnormalities in dogs affected by canine anaplasmosis were a thrombocytopenia and a lymphopenia; most cases of anaemia were non-regenerative. Schlüsselwörter Hund, A. phagocytophilum, hämatologische Veränderungen, Öster - reich Zusammenfassung Gegenstand und Ziel: Im Rahmen der Diagnostik der kaninen Anaplasmose in der Tierarztpraxis sollen Abweichungen hämatologischer Parameter, die Beurteilung eines Blutausstriches und die Berücksichtigung der saisonalen Bedingungen zur Diagnose einer Anaplasmose führen. Material und Methode: Es wurden 57 Blutproben von Hunden untersucht. Die Diagnose einer kaninen Anaplasmose wurde durch Untersuchung eines gefärbten Blutausstriches in allen 57 Fällen bestätigt. Ergebnisse: Bei 5 Hunden (9,%) wurde eine Thrombozytopenie nachgewiesen. Die Erythrozytenanzahl lag in 3,%, der Hämatokrit in,5% und der Hämoglobinwert in,3% der Fälle unterhalb des Referenzbereichs. Mittels Retikulozytenzählung wurde in 7,% der Fälle eine nicht regenerative Anämie diagnostiziert. Eine Leukopenie ergab sich zu 35,1%, eine Lymphopenie zu,7%, eine Neutropenie zu,9%, eine Monozytose zu 5,7% und eine Eosino - philie zu 15,%. Schlussfolgerung: Die Resultate zeigen, dass bei Hunden die Verdachtsdiagnose einer Anaplasmose aufgrund von typischen hämatologischen Veränderungen gestellt werden kann. Die wichtigsten Veränderungen im Blutbild der betroffenen Hunde waren eine Thrombozytopenie, eine Lymphopenie und im Fall einer vorliegenden Anämie in den meisten Fällen eine nicht regenerative Anämie. Correspondence to Georges Kirtz INVITRO Laboratory Rennweg 95 3 Vienna Austria georges.kirtz@invitro.at In-House-Diagnose der kaninen Anaplasmose unter Berücksichtigung von hämatologischen Veränderungen und der Beurteilung eines gefärbten Blut - ausstrichs Tierärztl Prax 15; 3 (K): 1 Received: May 19, 1 Accepted after revision: August, 1 Epub ahead of print: March, 15 Introduction Canine anaplasmosis is a tick-borne disease caused by Anaplasma phagocytophilum, an obligatory intracellular gram-negative pleomorphic bacterium. The primary target cells of this bacterium are circulating neutrophils (1). In Europe A. phagocytophilum is mainly transmitted by the hard tick Ixodes ricinus (). The first occurrence of inclusion bodies of A. phagocytophilum are usually seen between and 1 days post infection as. µm small elementary bodies or as up to µm large, characteristic inclusion bodies (morulae) in the cytoplasm of circulating neutrophils ( Fig. 1). The incubation period is 13 weeks (3). Affected dogs show mostly unspecific clinical signs like fever (rectal temperature up to 1 C), anorexia and depression. Other reported clinical signs include vomiting, petechiae, gingival bleeding, epistaxis, anaemia, reluctance to move, polyarthritis, splenomegaly, hepatomegaly, en- Schattauer 15 Tierärztliche Praxis Kleintiere /15

G. Kirtz; E. Leidinger: In-clinic diagnosis of canine anaplasmosis Materials and methods Fig. 1 Blood smear: elementary bodies (thin arrow) and Morulae (thick arrow) of A.

2 G. Kirtz; E. Leidinger: In-clinic diagnosis of canine anaplasmosis Materials and methods Fig. 1 Blood smear: elementary bodies (thin arrow) and Morulae (thick arrow) of A. phagocytophilum in two neutrophils in a dog s blood. Abb. 1 Blutausstrich: Elementarkörperchen (dünner Pfeil) und Morulae (dicker Pfeil) von A. phagocytophilum in zwei Neutrophilen im Blut eines Hundes larged lymph nodes and meningitis (7). The most common haematological findings in canine anaplasmosis are a normocytic, normochromic non-regenerative anaemia, thrombocytopenia, leukopenia, and occasionally leukocytosis with a left-shift (7, 15, 13, 1). The mechanism of the thrombocytopenia is not yet clear. Possible causes are disseminated intravascular coagulation with increased platelet consumption, sequestration in the enlarged spleen and/or an immune-mediated destruction of the platelets (1,, ). Another hypothesis is that some cells of the megakaryocytic lineage (MEG-1) are susceptible to an infection with A. phagocytophilum and in consequence of this infection an alteration in platelet production is induced (5). The thrombocyte count usually returns to the reference interval within a few days after bacteraemia or after successful therapy (13). A rapid method for the diagnosis of canine anaplasmosis is the microscopic evaluation of a blood smear. Elementary bodies or morulae of A. phagocytophilum are usually found within the cytoplasm of granulocytes in the periphery and in the feather-edge of the blood film ( Fig. 1), but only for a short time. Inclusion bodies appear as early as days after infection and persist for approximately days (). A further diagnostic tool is the molecular analysis (PCR) of blood samples for A. phagocytophilum. Single serological tests are not useful in the diagnosis of acute canine anaplasmosis because the seroprevalence in endemic areas is often high (, 9). The aim of this retrospective study was to show that haematological abnormalities, evaluation of a blood smear and seasonal variation may serve as helpful tools in the in-house diagnosis of the acute phase of canine anaplasmosis. In a retrospective study (13) a complete blood count by an automated analyser (CELL-DYN 35 with Veterinary Package Software, Abbott Laboratories, IL, USA) was performed on 57 canine blood samples from patients with clinical signs suspicious for canine anaplasmosis, or with fever of unclear origin as requested by veterinary practitioners. The examined dogs were between 7 months and 13 years old, belonging to 3 breeds. Twenty-nine dogs were entire males, six castrated males, four entire females, 1 neutered females, and in four dogs the sex was not reported. The majority of the dogs lived in or in the vicinity of regions known to be tick-infested areas in Eastern Austria. The analyses were performed in the private veterinary laboratory INVITRO, Vienna, Austria by two experienced haematologists. A microscopic -cell white blood cell differential (WBCD) was performed on stained blood smears (Hemacolor, Merck, Vienna, Austria). Samples with haemolysis or lipemia were not excluded from this retrospective study. The presence of A. phagocytophilum was confirmed by microscopic examination of a stained blood smear ( feather-edge and along both sides). As infection with Babesia canis canis is a possible differential diagnosis, all samples from animals included in the study were examined for Babesia canis microscopically, using the concentration line technique; however no positive cases were identified. A microscopic reticulocyte count was performed in the anaemic dogs as described previously (1) and the absolute values were calculated. The haematological evaluation included a complete blood count (CBC) and a WBCD. The CBC comprised the following parameters: red blood cell count (RBC), haemoglobin (HGB), packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC), leucocyte count (WBC), thrombocyte count and absolute reticulocyte count. WBCD included determination of relative and absolute values of neutrophils, lymphocytes, monocytes, eosinophils and basophils. The degree of anaemia (mild [PCV:.3.37 l/l], moderate [PCV:..9 l/l], severe [PCV: l/l] and very severe [PCV: <.13 l/l]), and of regeneration (none: reticulocytes up to x 9 /l, slight: up to 15 x 9 /l, moderate: up to 3 x 9 /l, marked: more than 5 x 9 /l) were classified as mentioned by Tvedten (1). Thrombocytopenia was classified as slight (15 x 9 /l), mild (995 x 9 /l), moderate (95 x 9 /l) and severe (< 5 x 9 /l) (). The standard deviation, median and arithmetical mean were calculated for all parameters, using standard mathematical formulae (Analyse-It, version 1.73 for Microsoft Excel, Analyse-It. software ldt). Results Thrombocytopenia was detected in 5 (9.%) dogs. In accor - dance with the classification mentioned above, a severe thrombo - Tierärztliche Praxis Kleintiere /15 Schattauer 15

3 G. Kirtz; E. Leidinger: In-clinic diagnosis of canine anaplasmosis 3 cytopenia was detected in dogs (17.%). The lowest measured value was x 9 /l. A moderate thrombocytopenia was seen in dogs (.1%), a mild thrombocytopenia in 1 dogs (.%), and a slight thrombocytopenia in six dogs (.5%). One dog had a thrombocyte count within the reference interval (151 x 9 /l). This value however, is close to the lower reference limit. The anaemia (PCV) was classified as mild in nine dogs (15.%), moderate in one (1.%), severe in two (3.%) and very severe in two dogs (3.%). In 3 dogs (3.%) PCV values were within and in seven dogs (1.3%) above the reference values. According to the classification mentioned above, analysis of the absolute reticulocyte counts in anaemic dogs showed the following results: eleven positive dogs (7.%) had no regeneration; in three dogs (1.%) a slight regeneration was noticed. The leucocyte count revealed a leukopenia in dogs (35.1%). Haematology results including the WBCD are displayed in Table 1 and Fig.. All dogs, with the exception of one dog found positive in February, were diagnosed between April and November. The majority (73.7%) of the A. phagocytophilum-positive dogs were diagnosed in the months of May and June ( Fig. 3). Discussion Anaplasmosis is a world-wide infectious disease reported in many species including dogs, cats, horses, ruminants and humans and Table 1 Haematological parameters in dogs with acute anaplasmosis. Hämatologische Parameter bei Hunden mit einer akuten Anaplas- Tab. 1 mose Parameter (reference values) Thrombocytes (155 x 9 /l) RBC (5.5. x 1 /l) PCV (.37.5 l/l) Haemoglobin (11 g/l) MCV (77 fl) MCHC (3135%) Reticulocytes (> x 9 /l) WBC (1 x /l) Neutrophils (3.39. x 9 /l) Bands (<.5 x 9 /l) Lymphocytes (1.3. x 9 /l) Monocytes (< 1. x 9 /l) Eosinophils (<.5 x 9 /l) Basophils (<.5 x 9 /l) Percentage of Anaplasma-positive samples compared to reference values below 9. 3., within above has gained rising attention in the last decades. A case of anaplasmosis in a timber wolf has recently been reported (). As clinical signs of acute canine anaplasmosis and other tick-borne diseases like acute babesiosis are often unspecific, veterinarians often request haematological analysis. A moderate to slight thrombocytopenia (.%) was the most common haematological abnormality in this retrospective study. This result is in accordance with other studies (, 13). Concerning the WBC a leukocyte count within the reference range was the most common finding here. A leukopenia was diagnosed in 35.1% of the patients. In the acute a b c Thrombocytes ( 9 /l) Reticulocytes ( 9 /l) < >3. >. >5. WBC ( 9 /l) Fig. Thrombocyte count (a), reticulocyte count (b) and white blood cells count (c) in A. phagocytophilum-positive dogs. Abb. Thrombozyten- (a), Retikulozyten- (b) und Leukozytenzahl (c) bei A.-phagocytophilum-positiven Hunden Schattauer 15 Tierärztliche Praxis Kleintiere /15

4 G. Kirtz; E. Leidinger: In-clinic diagnosis of canine anaplasmosis Jan. Feb. March April May June July Aug. Sept. Oct. Nov. Dec. Month Fig. 3 Seasonal occurrence of canine anaplasmosis. Abb. 3 Saisonales Vorkommen der kaninen Anaplasmose stage of anaplasmosis, and in acute babesiosis, there is often a decrease in leukocytes as a consequence of a severe consumption (). Lymphopenia (.7%) was the most common finding in the WBCD count (). Both neutropenia (.9%) and neutrophilia (1.3%) were present, although most of the affected dogs had neu - trophil values in the reference range. Less common abnormalities were monocytosis (5.7%), eosinophilia (15.%) and a left shift (13.%). These data are partially similar to those of another study (). Anaemia was present in only approximately a quarter of the affected dogs. These results are also in agreement with two previous studies (1, 15). The majority of the anaemic dogs showed a mild to moderate anaemia. The anaemia was found to be non-regenerative in the majority of cases (7.%). The lack of regeneration is due to the acute stage of the infection. After successful therapy or at the end of bacteraemia erythrocyte and PCV values return to the reference interval (1). The MCV was within the reference Conclusion for practice A major aspect of this retrospective study was that it was designed as an in-clinic study to assist veterinary practitioners with a rapid diagnosis of A. phagocytophilum based on a combination of clinical findings, haematological abnormalities and microscopic examination of a blood smear. The main clinical pathological findings in our study were a mild to moderate thrombocytopenia and a lymphopenia. When anaemia was present it was mild to moderate and usually non-regenerative. This study demonstrates that based upon haematological abnormalities in dogs during the acute phase of anaplasmosis, clinicians should perform further testing (blood smear evaluation) to confirm the tentative diagnosis in-house in a short time. Seasonal variations should also be taken into consideration when ranking canine anaplasmosis in a list of differentials in cases with appropriate clinical signs. range (normocytic) in most of our dogs, which is in accordance with the results of Kohn et al. (). MCHC values above the reference range were measured in 1% of our dogs. A possible reason for increased MCHC may be intravascular haemolysis. In this study however, preanalytical factors causing a preanalytical haemolysis (e. g. inappropriate collection techniques, inadequate tube filling, faulty sample transport) as well as sample lipemia are more probable causes of an increased MCHC. Although A. phagocytophilum-positive dogs were found from April to November, the majority of positive dogs were diagnosed in the early summer months of May and June. This is in accordance with other studies describing canine anaplasmosis (, 9) but in discordance with the appearance of another dangerous infectious agent, Babesia canis (, 11). Knowledge of these seasonal variations may be an important factor in the initial therapeutic approach for these two infections as both causative organisms often lead to similar clinical signs and haemotogical abnormalities. In contrast to acute babesiosis, where a severe thrombocytopenia is found in 55.% of affected dogs (), thrombocytopenia in acute anaplasmosis is more often moderate to slight (.%). The diagnostic criteria for confirmed canine anaplasmosis are clinical symptoms, haematogical abnormalities, and the detection of inclusion bodies of A. phagocytophilum within the neutrophils or a positive PCR (7) in combination with seasonal variations. In the acute phase of the disease where the above described haematological abnormalities are seen, inclusion bodies of A. phagocytophilum can usually be demonstrated in circulating granulocytes. However, this method depends on the quality of the smear as well as on the experience of the examiner, sometimes leading to sub - jective errors. But even if the typical inclusion bodies are not detected in the blood smear, the combination of clinical signs, haematological abnormalities, in particular thrombocytopenia and seasonal data should lead the veterinarian to a tentative diagnosis of anaplasmosis and initiation of the appropriate therapy. In cases of arguable blood smear results, especially in cases with low parasitaemia, PCR may be an alternative way to confirm the diagnosis. PCR is reported to have a higher sensitivity than the blood smear technique (), however reporting is frequently delayed and a the - rapy should be initiated before final results are known. In cases where morulae of A. phagocytophilum were detected in blood smears, veterinary practitioners often declined a PCR for financial reasons. In this retrospective study the authors cannot rule out that some cases with low parasitaemia may have been missed. But this unlikely would change the main results of this study. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of this paper. Tierärztliche Praxis Kleintiere /15 Schattauer 15

5 G. Kirtz; E. Leidinger: In-clinic diagnosis of canine anaplasmosis 5 References 1. Bexfield NH, Villiers EJ, Herrtage ME. Immune-mediated haemolytic anaemia and thrombocytopenia associated with Anaplasma phagocytophilum in a dog. J Small Anim Pract 5; : Carrade DD, Foley JE, Borjesson DL, Sykes JE. Canine granulocytic ana - plasmosis: a review. J Vet Intern Med 9; 3: Egenvall A, Bjöersdorff A, Lillihöök L, Olson-Engvall E, Karlstam E, Artursson K, Hedhammer A, Gunnarson A. Early manifestations of granulocytic ehrlichiosis in dogs inoculated experimentally with a Swedish Ehrlichia species isolate. Vet Rec 199; 13: Furlanello T, Fioro F, Caldin M, Lubas G, Solano-Gallego L. Clinicalpatho - logical findings in naturally occurring cases of babesiosis caused by large Babesia from dogs in northeastern Italy. Vet Parasitol 5; 13: Granik JL, Reneer DV, Carlyon JA, Bonesson DL. Anaplasma phagocytophilum infects cells of the megakaryocytic lineage through sialylated ligands but fails to alter platelet production. J Med Microbiol ; 57: 13.. Kirtz G, Leschnik M, Hooijberg E, Tichy A, Leidinger E. In-clinic laboratory diagnosis of canine babesiosis (babesia canis canis) for veterinary practitioners in Central Europe. Tierärztl Prax 1; (K): Kirtz G, Meli M, Leidinger E, Ludwig P, Thum D, Czettel B, Kölbl S, Lutz H. Anaplasma phagocytophilum infection in a dog: identifying the causative agent using PCR. J Small Anim Pract 5; : Kohn B, Galke D, Beelitz P, Pfister K. Clinical features of canine granulocytic anaplasmosis in 1 naturally infected dogs. J Vet Intern Med ; : Kohn B, Silaghi C, Galke D, Arndt G, Pfister K. Infections with Anaplasma phagocytophilum in dogs in Germany. Res Vet Sci 11; 91 (1): Leschnik M, Kirtz G, Virányi S, Wile-Piazzai W, Duscher G. Acute granulocytic anaplasmosis in a captive timber wolf (canis lupus occidentalis) J Zoo Wildl Med 1; 3 (3): Leschnik M, Kirtz G, Tichy A, Leidinger E. Seasonal occurrence of canine babesiosis is influenced by locate climate conditions. Int J Med Microbiol ; 9: (Suppl): Lillihöök I, Egenvall A, Tvedten HW. Hematopathology in dogs experimentally infected with a Swedish granulocytic Ehrlichia species. Vet Clin Pathol 199; 7: Ravnik U, Tozon N, Strasek Smrdel K, Zupanc Avsic T. Anaplasmosis in dogs: the relation of haematological, biochemical and clinical alterations to antibody titre and PCR confirmed infection. Vet Microbiol 11; 19: Schaarschmidt D, Müller W. Labordiagnostische und klinische Aspekte der kaninen Anaplasmose und Ehrlichiose. Tierärztl Prax 7; 35 (K): Wistrand Mazepa A, Benjamin Kidd L, Young KM, Trepanier LA. Clinical presentation of Anaplasma phagocytophilum-seropositive dogs residing in a endemic area. J Am Anim Hosp Assoc ; : Tvedten H. Erythrocyte disorders. In: Small Animal Clinical Diagnosis by Laboratory Methods, nd edn. Tvedten H, Turnwald GH, eds. Philadelphia: Saunders 199; Schattauer 15 Tierärztliche Praxis Kleintiere /15

Key words Dog, babesiosis, haematological abnormalities, climate conditions. Schlüsselwörter Hund, Babesiose, hämatologische Veränderungen, Klimadaten

Key words Dog, babesiosis, haematological abnormalities, climate conditions. Schlüsselwörter Hund, Babesiose, hämatologische Veränderungen, Klimadaten Original Article Schattauer 2012 87 In-clinic laboratory diagnosis of canine babesiosis (Babesia canis canis) for veterinary practitioners in Central Europe G. Kirtz 1 ; M. Leschnik 2 ; E. Hooijberg 1