Monoclonal Gammopathy Associated With Heartworm Disease in a Dog A 12-year-old, intact female, mixed Yorkshire terrier was evaluated for syncopal episodes, weakness, decreased appetite, and weight loss. Heartworm disease was diagnosed based on evidence of circulating microfilariae of Dirofilaria immitis on direct examination of blood smears and a positive SNAP heartworm antigen test. An immunoglobulin G (IgG) gammopathy, demonstrated by serum protein electrophoresis, was associated with heartworm disease in this dog. Response to treatment with both an adulticide and the microfilaricide ivermectin included remission of clinical signs and a decrease in the monoclonal gammopathy. To our knowledge, this is the first report of an IgG gammopathy associated with heartworm disease in the dog. J Am Anim Hosp Assoc 2009;45:296-300. Donato de Caprariis, DVM Mariateresa Sasanelli, DVM Paola Paradies, PhD, DVM Domenico Otranto, PhD, DVM, Diplomate EVPC, Fellow RES Riccardo Lia, DVM C From the Department of Veterinary Public Health and Animal Sciences, Faculty of Veterinary Medicine, University of Bari, Strada Provinciale per Casamassima, Km. 3, Valenzano, Bari, 70010 Italy. Introduction Heartworm disease caused by Dirofilaria (D.) immitis is a life-threatening infection in dogs that occurs throughout the temperate and tropical areas of the world, particularly the United States, Japan, Australia, the Mediterranean, and southern Europe. The typical signs of chronic coughing, dyspnea, exercise intolerance, syncope, hemoptysis, and congestive heart failure are produced by the disease mechanisms initiated by the adult heartworm parasite. 1 Case Report A 12-year-old, 6-kg, intact female, mixed Yorkshire terrier was presented with a 1-week history of syncopal episodes precipitated by paroxysms (bursts) of coughing and a 9-month history of exercise intolerance and cough. The referring veterinarian had previously auscultated a left apical systolic murmur (2/6 grade, Levine scale), and mitral valve insufficiency with left atrial enlargement was diagnosed on clinical and radiographic findings. The dog was treated with enalapril (0.5 mg/kg q 24 hours per os [PO]) and furosemide (1 mg/kg q 12 hours PO). A mild improvement was reported by the owner; however, over the last months, the owner reported the dog s persistent weakness, inappetence, and mild weight loss. At referral, the dog was thin (body condition score 3/9) and depressed. On physical examination, rectal temperature was found to be 38.2 C, and pale mucous membranes were evident. Prescapular and popliteal lymphadenopathy, weak femoral pulses, tachycardia (180 beats per minute), and tachypnea (60 breaths per minute) were also recorded. A left apical systolic murmur (5/6 grade) that radiated to the right side and increased bronchovesicular sounds were evident on auscultation. Hepatosplenomegaly was noted on abdominal palpation. Lateral and ventrodorsal thoracic radiographs demonstrated evidence of moderate enlargement of both left cardiac chambers, mild dilatation of the main pulmonary artery, and an enlarged right caudal pulmonary artery. A mild perihilar infiltrate and increased interstitial density of the caudal lung lobes were also noted. Echocardiography revealed mitral and 296 JOURNAL of the American Animal Hospital Association
November/December 2009, Vol. 45 Monoclonal Gammopathy Associated With Heartworm Disease 297 tricuspid valve insufficiency due to degenerative valvular disease. Abdominal ultrasonography revealed hepatomegaly with decreased echogenicity, and dilatation of the caudal vena cava and hepatic veins. In addition, the spleen was mildly enlarged and hypoechoic, and enlarged splenic veins were also evident, which suggested portal hypertension and/or congestion. Results of a complete blood count (CBC) revealed severe anemia and neutrophilic leukocytosis with a left shift [see Table]. The platelet count and mean platelet volume were normal. A marked regenerative anemia and persistent autoagglutination were noted. A moderate number of nucleated erythrocytes, many spherocytes, and few schistocytes were also detected on peripheral blood films. The hematological abnormalities were indicative of immune-mediated hemolytic anemia (IMHA). Circulating microfilariae of D. immitis were identified on direct examination of blood smears and by the modified Knott s test, and they were differentiated from D. repens and D. reconditum microfilaria by morphological criteria and measuring parasite length and width. A SNAP heartworm antigen test a was also positive. Urinalysis revealed a specific gravity of 1.030, ph of 6, proteinuria (2+), and bilirubinuria. The urine protein to creati- Table Main Hematological and Serum Protein Electrophoresis (SPE) Abnormalities, Observed at Various Time Points During Case Presentation for Evaluation of a Monogammaglobulinemia Secondary to Canine Heartworm Disease Test Presentation Day 7 Day 35 Day 66 Day 110 Reference Range CBC * Packed cell volume 18 21 25 26.1 29.6 37%-50% Corrected reticulocyte count 6.4 ND ND ND ND Red cell production index 3.2 ND ND ND ND Absolute reticulocyte count 390.4 ND ND ND ND 60-80 10 3 /µl Leukocytes 22.2 33.2 46.4 12.0 9.6 6-17 10 3 /µl Neutrophils 16.032 28.400 39.600 8.524 6.721 3.500-9.300/µL Bands 840 112 234 0 0 0-300/µL Lymphocytes 2.664 2.522 2.824 2.765 2.342 1.200-3.200/µL Eosinophils 1.110 670 445 354 256 100-800/µL Monocytes 1.554 1.520 3.381 412 343 200-1200/µL Platelets 184 220 205 195 255 180-450 10 3 /µl Mean platelet volume 9.2 10.4 10.8 9.7 8.5 8-12 fl SPE Total protein 7.5 ND 7.2 7.0 6.4 5.5-7.5 g/dl Albumin 2.17 2.4 2.5 2.6 2.4-4.0 g/dl Globulins 5.33 4.8 4.5 3.8 3.6-4.8 g/dl α 1 0.20 0.10 0.20 0.17 0.1-0.5 g/dl α 2 0.77 1.6 0.50 0.5 0.4-1.4 g/dl β 1.60 0.66 1.8 1.5 0.7-1.8 g/dl γ 2.77 2.44 2.0 1.63 0.7-1.7 g/dl * CBC=complete blood count ND=not done
298 JOURNAL of the American Animal Hospital Association November/December 2009, Vol. 45 nine ratio was within normal limits. The heat precipitation test for Bence Jones proteins was negative. Results of serum biochemical analysis revealed an increased serum alkaline phosphatase (250 IU/L, reference interval 35 to 180 IU/L); increased liver enzyme activity (aspartate aminotransferase 92 IU/L, reference interval 5 to 40 IU/L; alanine aminotransferase 175 IU/L, reference interval 5 to 35 IU/L); increased total bilirubin (0.6 mg/dl, reference interval 0 to 0.4 mg/dl); hypoalbuminemia; and hyperglobulinemia. Serum protein electrophoresis (SPE) showed a narrow spike in the γ region [Figure 1A]. The monoclonal protein was identified by SPE as immunoglobulin G (IgG). A prolonged prothrombin time and partial thromboplastin time, increased fibrin degradation products (36 µg/ml, reference interval <5 µg/ml), and decreased antithrombin activity (70% of normal) were suggestive of compensated disseminated intravascular coagulation (DIC). Serological tests were negative for Leishmania infantum and Ehrlichia canis. Lymph node and bone marrow aspirates were obtained and assessed by cytological examinations. Bone marrow aspirates were highly cellular with mild megakaryocytic and erythroid hyperplasia and mild plasmacytosis. Cytological evaluation of fine-needle aspirates of the popliteal nodes revealed a moderate grade of hyperplasia/reactivity, marked erythrophagocytosis, and increased numbers of well-differentiated plasma cells. Neither cellular evidence of lymphoid neoplasia nor amastigotes of Leishmania spp. were observed in lymph node and bone marrow aspirates. Based on the above described findings, a definitive diagnosis of heartworm disease with monoclonal gammopathy was made. Even if the dog did not appear to have a large worm burden, the risk was high for the development of thromboembolism after adulticide therapy because of the coagulation abnormalities and severe heartworm disease (i.e., the dog was considered class 2 according to information reported in Figures 1A-1D Sequential serum protein electrophoresis patterns from a 12-year-old, female, mixed Yorkshire terrier with heartworm disease. (A) Initial presentation. Note the monoclonal peak in the γ region, which represents 36.9% (2.77 g/dl) of the total serum protein concentration (7.5 g/dl). (B) Day 66 after initial presentation. Note the monoclonal peak in the γ region, which represents 28.5% (2.0 g/dl) of the total serum protein concentration (7.0 g/dl). (C) Day 110 after initial presentation. Note the monoclonal peak in the γ region, which represents 25.5% (1.63 g/dl) of the total serum protein concentration (6.4 g/dl). (D) Day 170 after initial presentation. Note the γ region, which represents 15.5% (1.12 g/dl) of the total serum protein concentration (7.2 g/dl).
November/December 2009, Vol. 45 Monoclonal Gammopathy Associated With Heartworm Disease 299 the Immiticide data sheet). b The dog was strictly confined in a cage and treated with prednisone (2 mg/kg q 24 hours PO) for IMHA. The enalapril dosage was increased (0.5 mg/kg q 12 hours PO), and furosemide was prescribed (1 mg/kg q 12 hours PO). On day 7 after presentation to our hospital, a mild improvement in the dog s general attitude was noted. The dog was alert and eating well, and coughing was reported during feeding. Results of CBC revealed a persistent anemia, mature neutrophilia, and a monocytosis. These changes were consistent with a chronic inflammatory leukogram. One dose of melarsomine (15 mg) into the muscle between the third and fifth lumbar vertebrae was administered, and the dog was observed for 48 hours after injection. Adverse reactions including anorexia, vomiting, and diarrhea occurred 24 hours following administration and were treated with supportive therapy. The dog was discharged 10 days after presentation, with owner instructions to provide cage rest and administer prednisone (0.5 mg/kg q 48 hours PO). On day 35 after the initial presentation, the dog returned for reevaluation. The owners reported that the attitude of the dog had improved gradually, but occasional episodes of coughing and heavy breathing persisted. On physical examination, the dog had pale mucous membranes, and increased bronchovesicular sounds were audible over the caudal dorsal lung lobes. The CBC revealed a persistent anemia, mature neutrophilia, and a monocytosis. Serum protein electrophoresis showed a persistent monoclonal peak in the γ region and an increased α 2 macroglobulin concentration. The standard protocol of adulticide therapy involving two injections of melarsomine 24 hours apart was subsequently instituted. The dog manifested depression, anorexia, and vomiting after the second dose of melarsomine. The dog was reevaluated 30 days following the second and third doses of adulticide administration (on day 66 after the initial presentation). The owner reported improvement in overall condition, and the dog had gained 0.5 kg (body condition score 4/9) and was only coughing intermittently. The CBC revealed a normocytic and normochromic anemia, and SPE showed a decrease in the monoclonal peak in the γ region [Figure 1B]. Prednisone was discontinued, and ivermectin was initiated at a dose of 5 µg/kg PO once a month to kill circulating microfilariae. The owner was instructed to start a prophylactic regimen with ivermectin once a month. On day 110 after initial presentation, the dog s clinical findings were unremarkable. The CBC revealed a normocytic and normochromic anemia, and SPE showed a marked reduction in the monoclonal peak in the γ region [Figure 1C]. On day 170 after initial presentation to our hospital, resolution of abnormal clinical signs and normalization of the CBC and hypergammaglobulinemia were evident [Figure 1D]. Discussion Heartworm disease due to D. immitis is a life-threatening infection in dogs that occurs worldwide throughout temperate and tropical areas. 2 Clinical signs of heartworm infection can be acute, but they are more commonly observed in chronically infected dogs and in dogs that manifest a marked allergic response to the adult worms and microfilariae. 3 Respiratory signs (i.e., coughing, tachypnea, dyspnea, hemoptysis), syncope, exercise intolerance, and ascites are the most common clinical signs. 4 The dog described in this report was diagnosed with heartworm disease based on the identification of D. immitis microfilaria and by detection of adult heartworm antigen in serum. Syncopal episodes precipitated by paroxysms of coughing were the primary complaint for the dog described in this study. In dogs with severe heartworm disease, syncope may occur secondary to exercise or excitement, and it may be explained by the combination of inadequate blood flow and decreased systemic vascular resistance leading to hypotension. 2 Syncopal episodes in the dog may also be related to a combination of decreased cerebral perfusion due to marked anemia, a severe increase in intracranial pressure during coughing, and/or an increased vagal tone mediated by pulmonary mechanoreceptors. 4,5 Heartworm infection commonly causes pathological lesions in pulmonary microvasculature and parenchyma, which leads to increased pulmonary vascular resistance and pulmonary hypertension. 2 The dog of this report had radiographic evidence of pulmonary hypertension caused by pulmonary arterial wall hypertrophy associated with heartworm disease. The dog also had a concurrent marked regenerative anemia that was consistent with IMHA. Although a Coombs test was not performed, anemia (packed cell volume <20%), persistent autoagglutination, marked spherocytosis, and leukocytosis with a left shift were highly supportive findings in the diagnosis of IMHA. Spherocytes can develop secondary to other causes, such as zinc toxicosis and rickettsial diseases. Other causes for regenerative anemia need to be considered, such as physical injury to erythrocyte membranes because of cell entanglement with heartworms, turbulent blood flow through a mass of heartworms, or incompetent heart valves. Only a few schistocytes were observed on blood smears, but the presence of DIC may have been responsible for erythrocyte injury resulting in shape changes to some spherical cells. Hyperglobulinemia is an expected feature of canine heartworm disease, but it is generally observed as a polyclonal gammopathy. 6 The dog in this case had hyperglobulinemia but not hyperproteinemia. In addition, a clonal gammopathy was demonstrated via SPE that identified the monoclonal protein as IgG. Dirofilariasis, similar to other inflammatory diseases, has been hypothesized to elicit atypical benign clonal proliferation of plasma cells. In this case, the parasite D. immitis may have been the initial stimulus for both the IMHA and monoclonal gammopathy. Monoclonal gammopathy suggests an excess production of immunoglobulin by clonal expansion of a single plasma cell. 7 A narrow spike occurring in the α 2, β, or γ-globulin region characterizes the electrophoretic pattern of a monoclonal gammopathy. In dogs, monoclonal gammopathies are
300 JOURNAL of the American Animal Hospital Association November/December 2009, Vol. 45 more frequently associated with multiple myeloma, macroglobulinemia, and lymphoproliferative tumors (e.g., lymphocytic and plasma cell neoplasias), of which chronic lymphocytic leukemia expresses paraproteinemia in >50% of cases. 8 Conditions associated with paraproteinemias in dogs also include cutaneous amyloidosis, plasmacytic gastroenterocolitis, and chronic infectious diseases (e.g., canine monocytic ehrlichiosis, leishmaniasis). 9 Most of the monoclonal gammopathies reported with canine ehrlichiosis have been reevaluated and are now classified as narrow polyclonal gammopathies. 10,11 The diagnostic evaluation of this dog was most consistent with D. immitis. The dog of this report lived in an area endemic for leishmaniasis and canine monocytic ehrlichiosis; 12 however, serological tests failed to demonstrate the presence of a specific antibody response. Conclusion A nonmyelomatous, monoclonal gammopathy represents an unusual finding in dirofilariasis, and resolution of clinical signs and normalization of hypergammaglobulinemia after heartworm therapy suggest the gammopathy in this case was associated with dirofilariasis. To our knowledge, this is the first report of a monoclonal gammopathy associated with heartworm disease. Footnotes a SNAP heartworm antigen test; Idexx Laboratories, Milano, Italy 20145 b Immiticide; Merial, Assago (MI), Italy 20090 References 11. Calvert CA, Rawlings CA. Diagnosis and management of canine heartworm disease. In: Kirk RW, ed. Current Veterinary Therapy VIII. Small Animal Practice. Philadelphia: Saunders, 1983:348-359. 12. Kittleson MD. Heartworm infestation and disease (Dirofilariasis). In: Kittleson MD, Kienle RD, eds. Small Animal Cardiovascular Medicine. St. Louis: Mosby, Inc., 1998:370-401. 13. Dunavent B, Keister DM, Tanner PA, et al. Correlation between heartworm disease classification, serum antigen concentration and associated clinical pathology parameters. In: Soll MD, Knight DH, eds. Proceedings of the Heartworm Symposium 95, Batavia, IL, 1995, American Heartworm Society. 14. Kittleson MD. Myxomatous atrioventricular valvular degeneration. In: Kittleson MD, Kienle RD, eds. Small Animal Cardiovascular Medicine. St. Louis: Mosby, Inc., 1998:297-318. 15. Knight DH. Heartworm infection. In: King LG, ed. Respiratory Disease in Dogs and Cats. Philadelphia: Saunders, 2004:517-525. 16. Willard MD, Tvedten H. Small Animal Clinical Diagnosis by Laboratory Methods. 4th ed. Philadelphia: Saunders, 2004:289-291. 17. Dorfman M, Dimski DS. Paraproteinemias in small animal medicine. Compend Contin Educ Pract Vet 1992;14(5):621-634. 18. MacEwen EG, Hurvitz AI. Diagnosis and management of monoclonal gammopathies. Vet Clin North Am 1977;7(1):119-132. 19. Font A, Closa JM, Mascort J. Monoclonal gammopathy in a dog with visceral leishmaniasis. J Vet Intern Med 1994;8(3):233-235. 10. Breitschwerdt EB, Woody BJ, Zerbe CA, et al. Monoclonal gammopathy associated with naturally occurring canine ehrlichiosis. J Vet Intern Med 1987;1(1):2-9. 11. Giraudel JM, Pagès JP, Guelfi JF. 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. 12. Buonavoglia D, Sagazio P, Gravino EA, et al. Serological evidence of Ehrlichia canis in dogs in southern Italy. New Microbiol 1995;18:83-86.