Clonality testing in the lymph nodes from dogs with lymphadenomegaly due to Leishmania infantum infection

Autoři: Antonio Melendez-Lazo aff001;  Anne-Katherine Jasensky aff001;  Ico Thais Jolly-Frahija aff004;  Alexandra Kehl aff001;  Elisabeth Müller aff001;  Ignacio Mesa-Sánchez aff004
Působiště autorů: LABOKLIN GmbH & Co. KG, Bad Kissingen, Germany aff001;  Laboratorio Veterinario LABOKLIN, Madrid, Spain aff002;  Hospital Veterinario Puchol, Madrid, Spain aff003;  Hospital Veterinario Valencia Sur, Silla, Spain aff004
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article



In southern European countries, multicentric lymphoma and leishmaniosis are the main differential diagnoses in dogs presented with generalized lymphadenomegaly. The cytological examination is in some cases inconclusive and polymerase chain reaction (PCR) for antigen receptor rearrangement (PARR) has become a common method to confirm or rule out a lymphoproliferative neoplasia. According to the literature, leishmaniosis may lead to clonal arrangements and therefore to a false diagnosis of lymphoma, but this assumption is made from a single leishmania infected dog. Therefore, the objective of this study was to prospectively evaluate results from PARR in dogs with lymphadenomegaly due to clinical leishmaniosis at the moment of diagnosis.

Materials and methods

31 dogs with a diagnosis of leishmaniosis based on the LeishVet guidelines were included in the study. Samples from enlarged lymph nodes were taken for cytological examination, clonality testing and Leishmania infantum PCR.


All 31 dogs had medium to high positive antibody titers against Leishmania spp. and 30/31 had a positive Leishmania PCR from the lymph node. A polyclonal arrangement for B cells (immunoglobulin heavy chain gene) and T cells (T-cell receptor gamma chain gene) antigen receptors was found in 28/31 dogs. Two out of 31 dogs showed a monoclonal arrangement for Ig with high (1:2) and low (1:7) polyclonal background respectively; and one of the 31 dogs showed a monoclonal arrangement for T cell receptor with low (1:3) polyclonal background.


Infections with Leishmania infantum resulted in clonal rearrangement, and therefore in a possible false diagnosis of lymphoma, in 3 out of 31 dogs (9.7%). Although, PARR is a useful method to differentiate lymphoma from reactive lymphoid hyperplasia in dogs with leishmaniosis, mono-/biclonal results should be interpreted carefully, especially in the presence of any degree of polyclonal background, and together with other clinicopathological findings.

Klíčová slova:

Dogs – Leishmania – Leishmaniasis – Lymph nodes – Lymphocytes – Lymphomas – Polymerase chain reaction – T cells


1. Solano-Gallego L, Miró G, Koutinas A, Cardoso L, Pennisi M, Ferrer L, et al. LeishVet guidelines for the practical management of canine leishmaniosis. Parasit Vectors. 2011;4: 86. doi: 10.1186/1756-3305-4-86 21599936

2. Naucke TJ, Lorentz S. First report of venereal and vertical transmission of canine leishmaniosis from naturally infected dogs in Germany. Parasit Vectors. 2012;5: 67. doi: 10.1186/1756-3305-5-67 22463789

3. Rosypal AC, Troy GC, Zajac AM, Frank G, Lindsay DS. Transplacental transmission of a North American isolate of Leishmania infantum in an experimentally infected beagle. J Parasitol. 2005;91: 970–972. doi: 10.1645/GE-483R.1 17089780

4. Naucke TJ, Amelung S, Lorentz S. First report of transmission of canine leishmaniosis through bite wounds from a naturally infected dog in Germany. Parasit Vectors. 2016;9: 256. doi: 10.1186/s13071-016-1551-0 27161343

5. de Freitas E, Melo MN, da Costa-Val AP, Michalick MSM. Transmission of Leishmania infantum via blood transfusion in dogs: Potential for infection and importance of clinical factors. Vet Parasitol. 2006;137: 159–167. doi: 10.1016/j.vetpar.2005.12.011 16414196

6. Dantas-Torres F, Brandão-Filho SP. Expansão geográfica da leishmaniose visceral no Estado de Pernambuco. Rev Soc Bras Med Trop. 2006;39: 352–356. doi: 10.1590/s0037-86822006000400007 17119750

7. Vaselek S, Ayhan N, Oguz G, Erisoz Kasap O, Savić S, Di Muccio T, et al. Sand fly and Leishmania spp. survey in Vojvodina (Serbia): first detection of Leishmania infantum DNA in sand flies and the first record of Phlebotomus (Transphlebotomus) mascittii Grassi, 1908. Parasit Vectors. 2017;10: 444. doi: 10.1186/s13071-017-2386-z 28950895

8. Svobodova V, Svoboda M, Friedlaenderova L, Drahotsky P, Bohacova E, Baneth G. Canine leishmaniosis in three consecutive generations of dogs in Czech Republic. Vet Parasitol. 2017;237: 122–124. doi: 10.1016/j.vetpar.2017.02.025 28279490

9. Silvestrini P, Batchelor D, Allenspach K, Maunder C, Seth M, Mas A, et al. Clinical leishmaniasis in dogs living in the UK. J Small Anim Pract. 2016;57: 453–458. doi: 10.1111/jsap.12503 27251904

10. Dumitrache MO, Nachum-Biala Y, Gilad M, Mircean V, Cazan CD, Mihalca AD, et al. The quest for canine leishmaniasis in Romania: the presence of an autochthonous focus with subclinical infections in an area where disease occurred. Parasit Vectors. 2016;9: 297. doi: 10.1186/s13071-016-1583-5 27209427

11. Kaszak I, Planellas M, Dworecka-Kaszak B. Canine leishmaniosis—an emerging disease. Ann Parasitol. 2015;61: 69–76. Available: 26342500

12. Shaw SE, Langton DA, Hillman TJ. Canine leishmaniosis in the United Kingdom: A zoonotic disease waiting for a vector? Vet Parasitol. 2009;163: 281–285. doi: 10.1016/j.vetpar.2009.03.025 19369005

13. Menn B, Lorentz S, Naucke TJ. Imported and travelling dogs as carriers of canine vector-borne pathogens in Germany. Parasit Vectors. 2010;3: 34. doi: 10.1186/1756-3305-3-34 20377872

14. Petersen CA, Barr SC. Canine Leishmaniasis in North America: Emerging or Newly Recognized? Vet Clin North Am Small Anim Pract. 2009;39: 1065–1074. doi: 10.1016/j.cvsm.2009.06.008 19932363

15. Duprey ZH, Steurer FJ, Rooney JA, Kirchhoff L V, Jackson JE, Rowton ED, et al. Canine visceral leishmaniasis, United States and Canada, 2000–2003. Emerg Infect Dis. 2006;12: 440–446. doi: 10.3201/eid1203.050811 16704782

16. Rosypal AC, Troy GC, Zajac AM, Duncan RB, Waki K, Chang KP, et al. Emergence of zoonotic canine leishmaniasis in the United States: isolation and immunohistochemical detection of Leishmania infantum from foxhounds from Virginia. J Eukaryot Microbiol. 2003;50 Suppl: 691–693. Available: doi: 10.1111/j.1550-7408.2003.tb00690.x 14736222

17. McKenna M, Attipa C, Tasker S, Augusto M. Leishmaniosis in a dog with no travel history outside of the UK. Vet Rec. 2019;184: 441–441. doi: 10.1136/vr.105157 30665949

18. Meléndez-Lazo A, Ordeix L, Planellas M, Pastor J, Solano-Gallego L. Clinicopathological findings in sick dogs naturally infected with Leishmania infantum: Comparison of five different clinical classification systems. Res Vet Sci. 2018;117: 18–27. doi: 10.1016/j.rvsc.2017.10.011 29153900

19. Noli C, Saridomichelakis MN. An update on the diagnosis and treatment of canine leishmaniosis caused by Leishmania infantum (syn. L. chagasi). Vet J. 2014;202: 425–435. doi: 10.1016/j.tvjl.2014.09.002 25266647

20. Maia C, Ramada J, Cristóvão JM, Gonçalves L, Campino L. Diagnosis of canine leishmaniasis: Conventional and molecular techniques using different tissues. Vet J. 2009;179: 142–144. doi: 10.1016/j.tvjl.2007.08.009 17936654

21. Maia C, Campino L. Methods for diagnosis of canine leishmaniasis and immune response to infection. Vet Parasitol. 2008;158: 274–287. doi: 10.1016/j.vetpar.2008.07.028 18789583

22. Solano-Gallego L, Cardoso L, Pennisi MG, Petersen C, Bourdeau P, Oliva G, et al. Diagnostic Challenges in the Era of Canine Leishmania infantum Vaccines. Trends Parasitol. 2017;33: 706–717. doi: 10.1016/ 28689776

23. Paltrinieri S, Gradoni L, Roura X, Zatelli A, Zini E. Laboratory tests for diagnosing and monitoring canine leishmaniasis. Vet Clin Pathol. 2016;45: 552–578. doi: 10.1111/vcp.12413 27805725

24. Saridomichelakis MN, Mylonakis ME, Leontides LS, Koutinas AF, Billinis C, Kontos VI. Evaluation of lymph node and bone marrow cytology in the diagnosis of canine leishmaniasis (Leishmania infantum) in symptomatic and asymptomatic dogs. Am J Trop Med Hyg. 2005;73: 82–86. 16014839

25. Mylonakis ME, Papaioannou N, Saridomichelakis MN, Koutinas AF, Billinis C, Kontos VI. Cytologic patterns of lymphadenopathy in dogs infected with Leishmania infantum. Vet Clin Pathol. 2005;34: 243–247. doi: 10.1111/j.1939-165x.2005.tb00048.x 16134072

26. Kaiser H. Animal neoplasia: a systemic review. In: Kaiser H, editor. Neoplasms: Comparative Pathology in Animals, Plants and Man. Baltimore: Williams & Wilkins; 1981.

27. Moulton J, Harvey J. Tumors of lymphoid and hematopoietic tissue. 3rd ed. In: Moulton J, editor. Tumors of domestic animals. 3rd ed. Berkeley, CA: University of California Press.; 1990. pp. 233–234.

28. Thomas R, Smith KC, Ostrander EA, Galibert F, Breen M. Chromosome aberrations in canine multicentric lymphomas detected with comparative genomic hybridisation and a panel of single locus probes. Br J Cancer. 2003;89: 1530–1537. doi: 10.1038/sj.bjc.6601275 14562028

29. Milman G, Smith KC, Erles K. Serological detection of Epstein-Barr virus infection in dogs and cats. Vet Microbiol. 2011. doi: 10.1016/j.vetmic.2010.12.013 21242039

30. Hayes HM, Tarone RE, Cantor KP, Jessen CR, McCurnin DM, Richardson RC. Case-Control Study of Canine Malignant Lymphoma: Positive Association With Dog Owner’s Use of 2, 4-Dichlorophenoxyacetic Acid Herbicides. JNCI J Natl Cancer Inst. 1991;83: 1226–1231. doi: 10.1093/jnci/83.17.1226 1870148

31. Keller ET. Immune-mediated disease as a risk factor for canine lymphoma. Cancer. 1992;70: 2334–2337. doi: 10.1002/1097-0142(19921101)70:9<2334::aid-cncr2820700920>;2-7 1394061

32. Foster AP, Sturgess CP, Gould DJ, Iwasaki T, Day MJ. Pemphigus foliaceus in association with systemic lupus erythematosus, and subsequent lymphoma in a cocker spaniel. J Small Anim Pract. 2000. doi: 10.1111/j.1748-5827.2000.tb03938.x 10879406

33. Ruslander DA, Gebhard DH, Tompkins MB, Grindem CB, Page RL. Immunophenotypic characterization of canine lymphoproliferative disorders. In Vivo. 1997;11: 169–72. Available: 9179611

34. Greenlee PG, Filippa DA, Quimby FW, Patnaik AK, Calvano SE, Matus RE, et al. Lymphomas in dogs. A morphologic, immunologic, and clinical study. Cancer. 1990;66: 480–490. doi: 10.1002/1097-0142(19900801)66:3<480::aid-cncr2820660314>;2-x 2364361

35. Wilkerson MJ, Dolce K, Koopman T, Shuman W, Chun R, Garrett L, et al. Lineage differentiation of canine lymphoma/leukemias and aberrant expression of CD molecules. Vet Immunol Immunopathol. 2005;106: 179–196. doi: 10.1016/j.vetimm.2005.02.020 15963817

36. Fournel-Fleury C, Magnol JP, Bricaire P, Marchal T, Chabanne L, Delverdier A, et al. Cytohistological and immunological classification of canine malignant lymphomas: comparison with human non-Hodgkin’s lymphomas. J Comp Pathol. 1997;117: 35–59. Available: doi: 10.1016/s0021-9975(97)80065-5 9263843

37. Waugh EM, Gallagher A, Haining H, Johnston PEJ, Marchesi F, Jarrett RF, et al. Optimisation and validation of a PCR for antigen receptor rearrangement (PARR) assay to detect clonality in canine lymphoid malignancies. Vet Immunol Immunopathol. 2016;182: 115–124. doi: 10.1016/j.vetimm.2016.10.008 27863542

38. Langner KF, Joetzke AE, Nerschbach V, Eberle N, Schuberth H-J, Koy M, et al. Detection of clonal antigen receptor gene rearrangement in dogs with lymphoma by real-time polymerase chain reaction and melting curve analysis. BMC Vet Res. 2014;10: 1. doi: 10.1186/1746-6148-10-1 24383544

39. Keller SM, Vernau W, Moore PF. Clonality Testing in Veterinary Medicine: A Review With Diagnostic Guidelines. Vet Pathol. 2016;53: 711–725. doi: 10.1177/0300985815626576 26933096

40. Burnett RC, Vernau W, Modiano JF, Olver CS, Moore PF, Avery AC. Diagnosis of Canine Lymphoid Neoplasia Using Clonal Rearrangements of Antigen Receptor Genes. Vet Pathol. 2003;40: 32–41. doi: 10.1354/vp.40-1-32 12627711

41. Langerak AW, Groenen PJTA, Brüggemann M, Beldjord K, Bellan C, Bonello L, et al. EuroClonality/BIOMED-2 guidelines for interpretation and reporting of Ig/TCR clonality testing in suspected lymphoproliferations. Leukemia. 2012. doi: 10.1038/leu.2012.246 22918122

42. Avery AC. Molecular Diagnostics of Hematologic Malignancies in Small Animals. Vet Clin North Am Small Anim Pract. 2012;42: 97–110. doi: 10.1016/j.cvsm.2011.11.001 22285160

43. Burkhard MJ, Bienzle D. Making Sense of Lymphoma Diagnostics in Small Animal Patients. Vet Clin North Am Small Anim Pract. 2013;43: 1331–1347. doi: 10.1016/j.cvsm.2013.07.004 24144094

44. Szczepański T, Pongers-Willemse MJ, Langerak AW, Harts WA, Wijkhuijs AJ, van Wering ER, et al. Ig heavy chain gene rearrangements in T-cell acute lymphoblastic leukemia exhibit predominant DH6-19 and DH7-27 gene usage, can result in complete V-D-J rearrangements, and are rare in T-cell receptor alpha beta lineage. Blood. 1999;93: 4079–4085. Available: 10361104

45. Szczepański T, Beishuizen A, Pongers-Willemse M, Hählen K, Van Wering E, Wijkhuijs A, et al. Cross-lineage T cell receptor gene rearrangements occur in more than ninety percent of childhood precursor-B acute lymphoblastic leukemias: alternative PCR targets for detection of minimal residual disease. Leukemia. 1999;13: 196–205. doi: 10.1038/sj.leu.2401277 10025893

46. Aresu L, Aricò A, Ferraresso S, Martini V, Comazzi S, Riondato F, et al. Minimal residual disease detection by flow cytometry and PARR in lymph node, peripheral blood and bone marrow, following treatment of dogs with diffuse large B-cell lymphoma. Vet J. 2014;200: 318–324. doi: 10.1016/j.tvjl.2014.03.006 24698669

47. Hiyoshi-Kanemoto S, Goto-Koshino Y, Fukushima K, Takahashi M, Kanemoto H, Uchida K, et al. Detection of circulating tumor cells using GeneScan analysis for antigen receptor gene rearrangements in canine lymphoma patients. J Vet Med Sci. 2016;78: 877–81. doi: 10.1292/jvms.15-0433 26888583

48. Brady SP, Magro CM, Diaz-Cano SJ, Wolfe HJ. Analysis of clonality of atypical cutaneous lymphoid infiltrates associated with drug therapy by PCR/DGGE. Hum Pathol. 1999;30: 130–136. Available: doi: 10.1016/s0046-8177(99)90266-6 10029439

49. Magro CM, Crowson AN, Kovatich AJ, Burns F. Drug-induced reversible lymphoid dyscrasia: A clonal lymphomatoid dermatitis of memory and activated T cells. Hum Pathol. 2003;34: 119–129. doi: 10.1053/hupa.2003.4 12612879

50. Alaibac M, Daga A, Harms G, Morris J, Yu RC, Zwingerberger K, et al. Molecular analysis of the gamma delta T-cell receptor repertoire in normal human skin and in Oriental cutaneous leishmaniasis. Exp Dermatol. 1993;2: 106–112. Available: doi: 10.1111/j.1600-0625.1993.tb00017.x 8162326

51. Gentilini F, Calzolari C, Turba ME, Bettini G, Famigli-Bergamini P. GeneScanning analysis of Ig/TCR gene rearrangements to detect clonality in canine lymphomas. Vet Immunol Immunopathol. 2009;127: 47–56. doi: 10.1016/j.vetimm.2008.09.014 18963006

52. Qurollo BA, Davenport AC, Sherbert BM, Grindem CB, Birkenheuer AJ, Breitschwerdt EB. Infection with Panola Mountain Ehrlichia sp. in a Dog with Atypical Lymphocytes and Clonal T-Cell Expansion. J Vet Intern Med. 2013;27: 1251–1255. doi: 10.1111/jvim.12148 23875820

53. Dobson JM, Samuel S, Milstein H, Rogers K, Wood JLN. Canine neoplasia in the UK: estimates of incidence rates from a population of insured dogs. J Small Anim Pract. 2002;43: 240–246. Available: doi: 10.1111/j.1748-5827.2002.tb00066.x 12074288

54. Giordano A, Paltrinieri S. Interpretation of capillary zone electrophoresis compared with cellulose acetate and agarose gel electrophoresis: reference intervals and diagnostic efficiency in dogs and cats. Vet Clin Pathol. 2010;39: 464–473. doi: 10.1111/j.1939-165X.2010.00255.x 21039711

55. Francino O, Altet L, Sánchez-Robert E, Rodriguez A, Solano-Gallego L, Alberola J, et al. Advantages of real-time PCR assay for diagnosis and monitoring of canine leishmaniosis. Vet Parasitol. 2006;137: 214–221. doi: 10.1016/j.vetpar.2006.01.011 16473467

56. Hwang M-H, Darzentas N, Bienzle D, Moore PF, Guscetti F, Morrison J, et al. A review of canine B cell clonality assays and primer set optimization using large-scale repertoire data. Vet Immunol Immunopathol. 2019;209: 45–52. doi: 10.1016/j.vetimm.2019.01.002 30885305

57. Hwang M-H, Darzentas N, Bienzle D, Moore PF, Morrison J, Keller SM. Characterization of the canine immunoglobulin heavy chain repertoire by next generation sequencing. Vet Immunol Immunopathol. 2018;202: 181–190. doi: 10.1016/j.vetimm.2018.07.002 30078594

58. Keller SM, Moore PF. A novel clonality assay for the assessment of canine T cell proliferations. Vet Immunol Immunopathol. 2012;145: 410–419. doi: 10.1016/j.vetimm.2011.12.019 22237398

59. van Dongen JJM, Langerak AW, Brüggemann M, Evans PAS, Hummel M, Lavender FL, et al. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: Report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia. 2003;17: 2257–2317. doi: 10.1038/sj.leu.2403202 14671650

60. Stokol T, Nickerson GA, Shuman M, Belcher N. Dogs with Acute Myeloid Leukemia Have Clonal Rearrangements in T and B Cell Receptors. Front Vet Sci. 2017;4: 76. doi: 10.3389/fvets.2017.00076 28620611

61. Good DJ, Gascoyne RD. Atypical lymphoid hyperplasia mimicking lymphoma. Hematol Oncol Clin North Am. 2009;23: 729–45. doi: 10.1016/j.hoc.2009.04.005 19577167

62. Magro CM, Crowson AN. Drug-induced immune dysregulation as a cause of atypical cutaneous lymphoid infiltrates: a hypothesis. Hum Pathol. 1996;27: 125–32. Available: doi: 10.1016/s0046-8177(96)90365-2 8617453

63. Quintana PG, Kapadia SB, Bahler DW, Johnson JT, Swerdlow SH. Salivary gland lymphoid infiltrates associated with lymphoepithelial lesions: A clinicopathologic, immunophenotypic, and genotypic study. Hum Pathol. 1997. doi: 10.1016/S0046-8177(97)90161-1

64. Raskin R. Hemolymphatic System. 3rd ed. In: Raskin R, Meyer D, editors. Canine and Feline Cytology: A Color Atlas and Interpretation Guide. 3rd ed. St. Louis, MO: Elsevier Inc; 2016. pp. 91–137.

65. Foglia Manzillo V, Pagano A, Guglielmino R, Gradoni L, Restucci B, Oliva G. Extranodal γδ-T-cell lymphoma in a dog with leishmaniasis. Vet Clin Pathol. 2008;37: 298–301. doi: 10.1111/j.1939-165X.2008.00048.x 18761522

66. Ferro S, Palmieri C, Cavicchioli L, Zan G De, Aresu L, Benali SL. Leishmania Amastigotes in Neoplastic Cells of 3 Nonhistiocytic Canine Tumors. Vet Pathol. 2013;50: 749–752. doi: 10.1177/0300985813480192 23482523

67. Burkitt MD, Duckworth CA, Williams JM, Pritchard DM. Helicobacter pylori-induced gastric pathology: insights from in vivo and ex vivo models. Dis Model Mech. 2017;10: 89–104. doi: 10.1242/dmm.027649 28151409

68. Beatty J. Viral causes of feline lymphoma: Retroviruses and beyond. Vet J. 2014;201: 174–180. doi: 10.1016/j.tvjl.2014.05.026 24928422

69. Murphy B, Eckstrand C, Castillo D, Poon A, Liepnieks M, Harmon K, et al. Multiple, Independent T Cell Lymphomas Arising in an Experimentally FIV-Infected Cat during the Terminal Stage of Infection. Viruses. 2018;10: 280. doi: 10.3390/v10060280 29794987

70. Kumar R, Daga MK, Kamble NL, Sothwal A, Singh T, Nayak HK, et al. Rare association of Visceral leishmaniasis with Hodgkin’s disease: A case report. Infect Agent Cancer. 2011;6: 17. doi: 10.1186/1750-9378-6-17 22011565

71. Brandonisio O, Spinelli R, Pepe M. Dendritic cells in Leishmania infection. Microbes Infect. 2004;6: 1402–1409. doi: 10.1016/j.micinf.2004.10.004 15596127

72. Font A, Closa JM, Mascort J. Monoclonal Gammopathy in a Dog With Visceral Leishmaniasis. J Vet Intern Med. 1994;8: 233–235. doi: 10.1111/j.1939-1676.1994.tb03223.x 8064662

73. Giraudel JM, Pagès J-P, Guelfi J-F. Monoclonal Gammopathies in the Dog: A Retrospective Study of 18 Cases (1986–1999) and Literature Review. J Am Anim Hosp Assoc. 2002;38: 135–147. doi: 10.5326/0380135 11908831

Článek vyšel v časopise


2019 Číslo 12
Nejčtenější tento týden