Signalment:  
Gross Description:  
Histopathologic Description:
Morphologic Diagnosis:  
1) Spleen, reactive hyperplasia, lymphoplasmacytic, associated with intraendothelial zoites, morphology consistent with R. vitalii.Â
2) Myocardium, myocarditis, lymphoplasmacytic mild to moderate, with mild fiber degeneration, associated with intraendothelial zoites, morphology consistent with R. vitalii.
Lab Results:  
The biochemistry panels showed mild increase in total plasma proteins due to increase in albumin, moderate increase in serum activity of alanine aminotransferase and marked increase in the total serum bilirubin (Table 2). Urinalysis revealed marked bilirubinuria.
Based on the laboratory results described above, a clinical diagnosis of extravascular hemolytic anemia was established. The marked spherocytosis suggested an immune mediated origin. The dog was treated with 1 mg/kg prednisone but died the following day.
Table 1 - CBC (reference values are within parentheses)
WBCs (/mm3) | 32200 | (6,000-17.000) | RBCs (x106/mm3) | 1.3 | (5.5-8.5) |
Neutrophils (%) | 72 | (60%-77%) | |||
Neutrophils (abs.) | 23184 | (3,000-11,500) | |||
Bands (%) | 6 | (0%-3%) | Hemoglobin (g/dL) | 3.8 | (12.0-18.0) |
Bands (abs.) | 1932 | (0-300) | |||
Metamyelocytes (%) | 2 | (0%) | |||
Metamyelocytes (abs.) | 644 | 0 | Hematocrit (%) | 12 | (37-55) |
Myelocytes (%) | - | (0%) | |||
Myelocytes (abs.) | - | 0 | |||
Lymphocytes (%) | 17 | (12%-30%) | MCV (fl) | 92.3 | (60.0-77.0) |
Lymphocytes (abs.) | 5474 | (1,000-4,800) | |||
Monocytes (%) | 2 | (3%-10%) | |||
Monocytes (abs.) | 644 | (150-1,350) | MCHC (%) | 31.7 | (32.0-36.0) |
Eosinophils (%) | 1 | (2%-10%) | |||
Eosinophils (abs.) | 322 | (100-1,250) | |||
Basophils (%) | - | (rare) | Platelets (x103/mm3) | 98 | (200-500) |
Basophils (abs.) | - | (rare) |
Table 2 - Biochemistry panel
Parameter | Unit | Result | Reference values |
Alanine aminotransferase | U/L | 180 | 4.0-24.0 |
Albumin | g/dL | 4.4 | 2.6-3.3 |
Creatinine | mg/dL | 1.2 | 0.5-1.5 |
Total bilirubin | mg/dL | 6.5 | 0.1-0.5 |
Fibrinogen | mg/dL | 200 | 200-400 |
Globulins | g/dL | 4.2 | 2.7-4.4 |
Total plasm proteins | g/dL | 8.8 | 6.0-8.0 |
BUN | mg/dL | 56.0 | 21.0-60.0 |
Condition:   Rangelia vitalii
Contributor Comment:  
It is currently accepted that R. vitalii is transmitted by tick vectors (Rhipicephalus sanguineus and Amblyomma aureolatum) to dogs and several wild mammal species in the State of Rio Grande do Sul (RS) in southern Brazil. The life cycle of R. vitalii is unknown, but it has been speculated that the vectors R. sanguineus and A. aureolatum circulate the protozoan between wild mammals and domestic dogs, the latter probably being an aberrant host.(3) Experimental transmission to susceptible dogs using blood from spontaneously affected dogs was achieved recently in two independent studies.(4,5) The clinical and laboratory aspects of experimental disease differ somewhat from natural disease, suggesting that the parasite needs to replicate in the tick in order to acquire some aspects of virulence. Experimental disease is also fatal if left untreated.
The great majority of dogs with spontaneous rangeliosis develop clinical signs of extravascular hemolysis: pallor of mucous membranes; icterus; and hepatosplenomegaly.(3,4) Other clinical signs include apathy, anorexia, fever, vomiting, diarrhea, mucopurulent oculonasal discharge, tachypnea, tachycardia, subcutaneous edema of the pelvic limbs, and petechiae and ecchymosis on the mucous membranes.(3)
Hematologic findings are typical of extravascular hemolysis and include hypochromic macrocytic anemia with excessive regeneration. Anisocytosis, polychromasia, Howell-Jolly bodies and several nucleated red blood cell precursors (metarubricytes and rubricytes) are observed on peripheral blood smears.(3,4) Most of the affected dogs also present varying degrees of spherocytosis,(3,4) a hematological finding highly suggestive of immune mediated hemolytic anemia.(1) The numbers of circulating reticulocytes are high (5%-28%, average 12,5%). Erythrophagocytosis is occasionally observed, particularly in those cases in which there is marked associated spherocytosis. Normochromic normocytic anemia is observed on occasion due to the extreme contrast between the small and falsely hypochromic-appearing spherocytes and the large polychromatophils recently released from the bone marrow. Plasma from affected dogs is bright yellow. WBC counts frequently reveals leucocytosis due to regenerative left shift resulting from long-standing and non-specific stimulation on the bone marrow. In some of the cases the leucocytosis may present as a leukemoid reaction. Other common hematological findings include lymphocytosis and monocytosis.(3)
In a smaller percentage of the cases of spontaneous canine rangeliosis, affected dogs develop a bleeding disorder similar to DIC characterized by extensive hemorrhage from the tips, margins, and outer surface of the pinnae.(5) Dogs affected in this manner have a moderate decrease in platelet numbers; numerous large platelets are observed in the circulation, indicating regenerative thrombocytopenia. However, due to its moderate intensity, it is apparent that thrombocytopenia itself is insufficient to induce the hemorrhage in these dogs, and some other disorder(s) of hemostasis may be in place.(3,4)
There is no specific biochemical test for the diagnosis of rangeliosis, but serum alanine aminotransferase (ALT) is increased in most cases. This elevation most likely results from anemia, hypoxia of centrolobular hepatocytes, and death of these cells. Bilirubin levels are consistently increased due to the extravascular hemolysis and the urine is darkened by the excretion of large amounts of bilirubin and urobilinogen; hemoglobinuria is never part of the clinical picture.(3)
In contrast to most infectious hemolytic anemias, the clinical diagnosis of rangeliosis is generally made on response to therapy, since the parasite is seldom found in the blood smears from affected dogs (only in approximately 4% of the cases).(3) Currently, there is no commercial or in-house tests to detect antibodies or antigens associated with R. vitalii. Fine needle aspiration (FNA) or excisional biopsy of lymph nodes, spleen and bone marrow with subsequent cytological evaluation can also be important aids in the clinical diagnosis. Due to the intraendothelial location of the parasite, fine needle aspiration may yield false negative results.
Necropsy findings in dogs dying from rangeliosis are consistent with those seen in other causes of extravascular hemolysis. The mucous membranes, subcutaneous tissue, muscle fascia, serosal surfaces and arterial intima are markedly icteric and peripheral blood is thin and watery. The liver is enlarged and has a red-orange hue or, in more severe cases, a greenish discoloration. Accentuation of the hepatic lobular pattern is common. The spleen is enlarged and fleshy and all lymph nodes are swollen, moist, and red,(3) and may have multifocal to coalescing white areas.(5) Hyperplastic bone marrow is markedly red and fills the entire marrow space.
Microscopic evaluation of lymph nodes reveals marked erythrophagocytosis and, depending on the stage of the disease, there is hemosiderosis and severe paracortical lymphoid hyperplasia. In the spleen, there is lymphoid hyperplasia characterized by marked plasmacytosis which, in some cases, resembles the plasma cell proliferation pattern observed in myelomas; however, in rangeliosis, most plasma cells are mature and there are few plasmablasts, Mott cells and/or flame cells.(3)
In non-lymphoid organs, the same lymphoplasmacytic infiltrates are observed, and occasionally there is a granulomatous infiltrate which includes multinucleate giant cells that sometimes phagocytize parasitized endothelial cells.(4) In the liver, there is centrolibular coagulative necrosis and accumulation of bile pigment. In the bone marrow, there is marked erythroid and megakaryocytic hyperplasia characterized by replacement of the marrow adipose tissue by hematopoietic cells with high mitotic index, a drop in the myeloid to erythroid ratio, and increased numbers of megakaryocytes and megakaryoblasts. Foci of extramedullary hematopoiesis can be observed frequently in the spleen and liver and less frequently in the lymph nodes and adrenal glands.(3)
The parasite is found within the cytoplasm of endothelial cells in several organs and consists of 2.0-2.5 μm round to oval, basophilic organism when stained with hematoxylin and eosin; its cytoplasm is pale and inconspicuous and the nucleus is prominent, basophilic and eccentrically located.3,5 The frequency in which the parasite was observed in several organs was reported from the study of 11 cases of canine rangeliosis as: kidneys (7/11), lymph nodes (7/11), liver (6/11), bone marrow (4/11), spleen (3/11), tonsils (2/11), lung, brain, stomach and gallbladder (1/11).(4) The authors pointed out that not all organs were histologically examined in each of the 11 cases. Other reports indicate lymph nodes, bone marrow, kidneys and choroid plexus are the organs with the highest parasite load.(4) About 20-30 organisms can be found within the cytoplasm of each parasitized endothelial cell. The organisms usually can be seen in smears from bone marrow sampled during the necropsy and stained with Giemsa and Panoptic.(4) In various reports, immunostaining for Leishmania chagasi, Neospora caninum and Toxoplasma gondii was consistently negative;(4,5) however, the parasite reacted positively with an anti-Babesia microti antibody.(5) R. vitalii was also positive on in situ hybridization for B. microti(4) indicating that the organism belongs to the same group as Babesia
However, unlike babesia R. vitalii is characterized by an intraendothelial stage. Parasitized cells are immunopositive for Willebrand factor, indicating their endothelial nature. The ultrastructural characteristics of R. vitalii were reported by Loretti & Barros 2005 (these authors mistakenly spelled the parasites name as R. vitalli) as having an apical complex that includes a polar ring and rhoptries but no conoid; the parasite is contained within a parasitophorous vacuole that had a trilaminar membrane with villar protrusions and was located within the cytoplasm of capillary endothelial cells.(6)
Necropsy finding of dogs dying from rangeliosis, coupled with the hematological findings, suggest the diagnosis of hemolytic anemia; the finding of the causative agent in the cytoplasm of endothelial cells confirms a presumptive diagnosis.
Differentials for this case should include infection by R. vitalii, Histoplasma capsulatum, Trypanosoma cruzi, Leishmania spp., Toxoplasma gondii, and Neospora caninum. Histoplasma and Leishmania organisms are typically found in macrophages and may elicit an intense histiocytic to granulomatous response. H. capsulatum yeasts stain with PAS and GMS stains. Leishmania and T. cruzi have a kinetoplast, which is absent in R. vitalii. Additionally, the amastigotes of T. cruzi form pseudocysts within the sarcoplasm of cardiomyocytes and not within endothelial cells. The zoites of T. gondii and N. caninum form 2-5 μm and 4-7 μm tachyzoites, respectively, and have no kinetoplast. N. caninum can parasitize endothelial cells but usually parasitize a large spectrum of host cells and are PAS positive. Similarly, T. gondii affects a large spectrum of host cells, although usually not endothelial cells, and induces a whole different set of lesions.
JPC Diagnosis:  
1. Heart: Myocarditis, lymphoplasmacytic and histiocytic, multifocal, mild to moderate with cardiomyocyte degeneration, capillary fibrin thrombi and numerous intraendothelial protozoa.
2. Spleen: Plasmacytosis, diffuse, marked with multifocal reticuloendothelial cell hyperplasia and scattered intraendothelial protozoa.
Conference Comment:  
While reviewing the submitted CBC, biochemistry panel and cytology, conference participants discussed the clinicopathologic differentiation between extravascular and intravascular hemolysis. The diagnostic features for each are briefly summarized in the chart below.(2)
Extravascular Hemolysis | Intravascular Hemolysis | |
Cause (general categories) |
1. Antibody and/or complement mediated 2. Decreased RBC deformability 3. Premature RBC aging (decreased glycolysis and [ATP]) 4. Increased macrophage phagocytosis |
1. Complement-mediated lysis 2. Physical damage 3. Oxidative damage 4. Osmotic lysis 5. Membrane alterations |
Onset | Usually chronic course with insidious onset | Peracute to acute |
CBC | Neutrophilia, monocytosis, thrombocytosis | No significant findings |
Biochemistry | Hyperbilirubinemia with the unconjugated form dominating early |
1. Hemoglobinemia: plasma discolored red;
increased MCHC and MCH; decreased serum
haptoglobin 2. Hyperbilirubinemia with unconjugated dominating early |
Urinalysis | Bilirubinuria | Hemoglobinuria; hemosiderinuria; -� Bilirubinuria |
Erythrocyte morphology | Spherocytes, schistocytes, keratocytes; if a regenerative response, reticulocytes, Howell- Jolly bodies, polychromasia and macrocytosis | Schistocytes, keratocytes, Heinz bodies, eccentrocytes |
Certain breeds of dogs and cats possess hereditary biochemical deficiencies or predispositions to extravascular hemolysis.(2) In dogs, phosphofructokinase deficiency is seen in cocker spaniels, English springer spaniels and some mixed breeds. Pyruvate kinase deficiency is noted to occur in the Basenji, beagle, Chihuahua, dachshund, pug, miniature poodle, West Highland White terrier, and Cairn terriers as well as Abyssinian, Somali and domestic shorthaired cats. A hereditary predisposition to hemolytic anemia is suspected in the border collie, cocker spaniel, English springer spaniel, German shepherd dog, Irish setter, Old English sheepdog, poodle and the whippet.
References:
2. Brockus CW, Andreasen CB. Erythrocytes. In: Latimer KS, Mahaffey EA, Prasse KW, eds. Duncan and Prasses Veterinary Laboratory Medicine: Clinical Pathology. 4th ed. Ames, IA: Blackwell Publishing; 2003:26-38.
3. Fighera RA: Rangeliose. Acta Scientiae Veterinariae 2007;35(Supl2):264-266.
4. Krauspenhar C, Fighera RA, Gra+�-�a DL. Anemia hemol+�-�tica em c+�-�es associada a protozo+�-�rios. MEDVEP 2008;1:273-281.
5. Loretti A, Barros SS. Hemorrhagic disease in dogs infected with an unclassified intraendothelial piroplasm in southern Brazil. Vet Parasitol. 2005;134:193-213.
6. Loretti A, Barros SS: Infec+�-�+�-�o por Rangelia vitalli (Nambiuv+�°, Peste de Sangue) em caninos: revis+�-�o. MEDVEP. 2004;2:128-144.