Chirek, Aleksandra

Kohn, Barbara

Liebisch, Gabriele

Nolte, Ingo

Pfister, Kurt

Silaghi, Cornelia

Thiel, Claudia

PubMed

Institutional Repository of the Freie Universität Berlin

  Published Ahead of Print 19 October 2011. 10.1128/JCM.06041-11. 2011, 49(12):4413. DOI:J. Clin. Microbiol. Thiel, Ingo Nolte, Gabriele Liebisch and Kurt PfisterCornelia Silaghi, Barbara Kohn, Aleksandra Chirek, Claudia Involved in Natural Infections of Dogs Findings on Anaplasma phagocytophilum Relationship of Molecular and Clinicalhttp://jcm.asm.org/content/49/12/4413Updated information and services can be found at: These include:REFERENCEShttp://jcm.asm.org/content/49/12/4413#ref-list-1This article cites 7 articles, 1 of which can be accessed free at: CONTENT ALERTS more»articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when newhttp://journals.asm.org/site/misc/reprints.xhtmlInformation about commercial reprint orders: http://journals.asm.org/site/subscriptions/To subscribe to to another ASM Journal go to:  on May 6, 2014 by FU Berlin, Univ.-Klinikum Benjamin Franklinhttp://jcm.asm.org/Downloaded from  on May 6, 2014 by FU Berlin, Univ.-Klinikum Benjamin Franklinhttp://jcm.asm.org/Downloaded from JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 2011, p. 4413–4414 Vol. 49, No. 120095-1137/11/$12.00 doi:10.1128/JCM.06041-11Copyright © 2011, American Society for Microbiology. All Rights Reserved.Relationship of Molecular and Clinical Findings on Anaplasma phagocytophilumInvolved in Natural Infections of DogsClinical manifestations of canine granulocytic anaplasmo-sis (CGA), caused by Anaplasma phagocytophilum—an ob-ligate intracellular tick-transmitted bacterium infecting neu-trophilic granulocytes—range from self-limiting to severe.Commonly, lethargy, inappetence, and fever and, further,lameness, pale mucous membranes, tense abdomen, diar-rhea, vomiting, surface bleeding, tachypnea, enlarged lymphnodes, and splenomegaly are observed. Laboratory abnor-malities may include thrombocytopenia, anemia, lymphope-nia, neutrophilia, neutropenia, leukocytosis, leukopenia,monocytosis, lymphocytosis, hypoalbuminemia, hyperglobu-linemia, increased liver enzymes, and hyperbilirubinemia.Subclinical infections without clinical signs are also possible(1, 2). A. phagocytophilum also causes disease in horses,cattle, sheep, and humans and is found in a large number ofpotential wild reservoir hosts (7). It shows genetic hetero-geneity, e.g., when isolated from different host species orgeographic origins (4, 5). No significant differences wereobserved in previous studies concerning the rates of sero-positivity or PCR positivity in dogs with and without clinicalsigns of CGA (3). We hypothesized that different 16S rRNAgene variants of A. phagocytophilum may be involved innatural infection of dogs, as has been shown in naturalinfection of horses (6) and that those might cause differentclinical outcomes. Thus, DNA was extracted with theQiagen DNA Minikit (Qiagen, Hilden, Germany) accordingto the manufacturer’s instruction from 66 frozen A. phago-cytophilum-positive blood samples from dogs originating inGermany (n  64), Sweden (n  1), and Switzerland (n 1), which were diagnosed as positive for A. phagocytophilumin routine diagnostic procedures or epidemiological screen-ings from 2003 to 2010. The full clinical data were available,and coinfection with other vector-borne infections was ex-cluded by PCR (data not shown). A nested PCR targetingthe partial 16S rRNA gene (497 bp), followed by sequenc-ing, was performed as previously described (5). Most fre-quently obtained was variant B (n  37; the amplified partequals the prototype sequence of the human granulocyticanaplasmosis agent [GenBank accession no. U02521]), fol-lowed by variant A (n  23; equals Frankonia 2 [accessionno. AF136712]). In 6 dogs, 5 other variants were detected(one dog was infected with two variants simultaneously), butthose were not included in the comparative analysis(FJ829788, FJ829789, JN656381, JN656383, FJ829790,FJ829791). Selected clinical or laboratory abnormalitieswere investigated concerning their relationship to sequencevariants A and B using Fisher’s exact test (GraphPad Prism5). Statistically significant differences (P  0.05) were ob-served for fever, lethargy, inappetence, thrombocytopenia,and lymphopenia, which all appeared more frequently withvariant B than with variant A, even though both variantscaused clinical disease (Table 1). These clinical and labora-tory abnormalities often appear together in CGA. Openquestions remain. Is variant A less pathogenic for dogs, ordid the acute phase of the disease pass unnoticed? Were thesubclinical dogs in this study persistently infected? It is notyet clear whether the results indicate that partial 16S rRNAgene variants A and B are expressions of different strains ofA. phagocytophilum with different pathogenicities for dogs.Further characterization of the variants is thus warranted,and variants should be studied further in in vitro and in vivoexperiments.TABLE 1. Comparative analysis of selected clinical and laboratoryparameters and A. phagocytophilum 16S rRNA gene variantsA and B occurring in natural infections of 60 dogsInvestigatedparameter andoccurrenceNo. of cases withsequence variant:Significantdifference(P value)Ah BiFeverYes 5 26 Yes (0.0001)No 16 11LethargyYes 10 30 Yes (0.0037)No 12 6InappetenceYes 8 25 Yes (0.0166)No 14 11LamenessYes 6 8 NoNo 16 30SplenomegalyYes 7 17 NoNo 3 11ThrombocytopeniaaYes 11 28 Yes (0.0226)No 11 7LeukopeniabYes 0 6 NoNo 22 28LymphopeniacYes 2 18 Yes (0.0265)No 8 10AnemiadYes 7 18 NoNo 15 17MonocytosiseYes 6 14 NoNo 4 16Increased ALP valuefYes 5 21 NoNo 10 10HypoalbuminemiagYes 4 13 NoNo 9 17a Defined as a platelet count of 180,000/l.b Defined as a white blood cell count of 6,000/l.c Defined as a lymphocyte count of 1,000/l.d Defined as a hematocrit of 0.40 mmol/liter.e Defined as a monocyte count of 500/l.f Defined as an alkaline phosphatase concentration of 97 IU/liter.g Defined as an albumin concentration of 28 g/liter.h GenBank accession no. FJ829748 to FJ829750, FJ829752, FJ829753, FJ829755,FJ829756, FJ829758 to FJ829762, JN656336 to JN656340, and JN656342 to JN656346.i GenBank accession no. FJ829764 to FJ829766, FJ829768, FJ829770,FJ829772 to FJ829774, FJ829777 to FJ829779, FJ829781 to FJ829784, FJ829786,JN656352 to JN656371, JN656378, and JN656380.4413 on May 6, 2014 by FU Berlin, Univ.-Klinikum Benjamin Franklinhttp://jcm.asm.org/Downloaded from We have no conflict of interest to declare.Thanks to Daniela Galke (Clinic of Small Animals, Faculty ofVeterinary Medicine, Freie Universita¨t Berlin, Berlin, Germany)for acquiring clinical data and Monia Mahling (Institute for Statis-tics, Ludwig-Maximilians-Universita¨t Mu¨nchen, Munich, Germany)for valuable advice concerning the statistical analysis.REFERENCES1. Carrade, D. D., J. E. Foley, D. L. Borjesson, and J. E. Sykes. 2009. Caninegranulocytic anaplasmosis: a review. J. Vet. Intern. Med. 23:1129–1141.2. Kohn, B., D. Galke, P. Beelitz, and K. Pfister. 2008. Clinical features ofCanine Granulocytic Anaplasmosis in 18 naturally infected dogs. J. Vet.Intern. Med. 22:1289–1295.3. Kohn, B., C. Silaghi, D. Galke, G. Arndt, and K. Pfister. 2011. Infections withAnaplasma phagocytophilum in dogs in Germany. Res. Vet. Sci. 91:71–76.4. Scharf, W., et al. 2011. Distinct host species correlate with Anaplasma phago-cytophilum ankA gene clusters. J. Clin. Microbiol. 49:790–796.5. Silaghi, C., et al. 2011. Genetic variants of Anaplasma phagocytophilum in wildcaprine and cervid ungulates from the Alps in Tyrol, Austria. Vector BorneZoonotic Dis. 11:355–362.6. Silaghi, C., G. Liebisch, and K. Pfister. 2011. Genetic variants of Anaplasmaphagocytophilum from 14 equine granulocytic anaplasmosis cases. Parasit.Vectors 4:161.7. Woldehiwet, Z. 2010. The natural history of Anaplasma phagocytophilum. Vet.Parasitol. 167:108–122.Cornelia Silaghi*Comparative Tropical Medicine and ParasitologyLudwig-Maximilians-Universita¨t Mu¨nchenMunich, GermanyBarbara KohnClinic of Small AnimalsFaculty of Veterinary MedicineFreie Universita¨t BerlinBerlin, GermanyAleksandra ChirekClinic of Small AnimalsFaculty of Veterinary MedicineFreie Universita¨t BerlinBerlin, GermanyClaudia ThielComparative Tropical Medicine and ParasitologyLudwig-Maximilians-Universita¨t Mu¨nchenMunich, GermanyIngo NolteSmall Animal ClinicUniversity of Veterinary MedicineHannover, GermanyGabriele LiebischZecklabBurgwedel, GermanyKurt PfisterComparative Tropical Medicine and ParasitologyLudwig-Maximilians-Universita¨t Mu¨nchenMunich, Germany*Phone: 49-89 2180 3622Fax: 49-89 2180 3623E-mail: cornelia.silaghi@tropa.vetmed.uni-muenchen.de Published ahead of print on 19 October 2011.4414 LETTERS TO THE EDITOR J. CLIN. MICROBIOL. on May 6, 2014 by FU Berlin, Univ.-Klinikum Benjamin Franklinhttp://jcm.asm.org/Downloaded from 

Relationship of molecular and clinical findings on Anaplasma phagocytophilum
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Relationship of molecular and clinical findings on Anaplasma phagocytophilum involved in natural infections of dogs

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