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Experimental acute canine monocytic ehrlichiosis: clinicopathological and immunopathological findings
Veterinary Parasitology 119 (2004) 73–86
Experimental acute canine monocytic ehrlichiosis: clinicopathological and immunopathological findings Márcio Botelho de Castro a,b , Rosangela Zacarias Machado b,∗ , Lucia Padilha Cury Tomaz de Aquino a,b , Antonio Carlos Alessi b , Mirela Tinucci Costa c a
Universidade de Franca, Av. Dr. Armando Salles de Oliveira, 201, CEP 14404-600 Franca, SP, Brazil b Department of Veterinary Pathology, Universidade Estadual Paulista (UNESP), Via de Acesso Prof. Paulo Donato Castellani, sem no. CEP 14870-000 Jaboticabal, SP, Brazil c Veterinary Teaching Hospital, Universidade Estadual Paulista (UNESP), Via de Acesso Prof. Paulo Donato Castellani, sem no., CEP 14870-000 Jaboticabal, SP, Brazil Received 1 September 2002; received in revised form 25 September 2003; accepted 16 October 2003
Abstract Clinical signs, humoral and cellular immune responses, and microscopic and gross tissue alterations resulting from acute experimental Ehrlichia canis infection in dogs were studied. Four dogs were inoculated with E. canis and four were used as uninfected controls. After a 10–14-day incubation period, infected dogs developed pyrexia up to 41 ◦ C for 6–8 days. Antibody titers to E. canis antigen were demonstrable in all inoculated dogs at 30 days post-infection. Necropsy of infected animals revealed pale mucous membranes, generalized lymphadenopathy, splenomegaly, edema and ascites. Microcopically, the main lesions were: lymphoreticular hyperplasia in cortical areas of lymph nodes and spleenic white pulp, periportal accumulation of mononuclear cells and centrolobular fatty degeneration of the liver. Kidneys presented with glomerulonephritis characterized by interstitial mononuclear infiltration. Immunophenotyping of lymphocytes from lymph nodes and spleen sections displayed alterations in IgG, IgM, CD3+ and CD8+ cells population in infected dogs. © 2003 Elsevier B.V. All rights reserved. Keywords: Ehrlichia canis; Ehrlichiosis; Dog; Immunopathology; Immunohistochemistry
∗ Corresponding author. Tel.: +55-16-32092664; fax: +55-16-32024275. E-mail address: [email protected] (R.Z. Machado).
0304-4017/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2003.10.012
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1. Introduction Canine monocytic ehrlichiosis (CME) is an infectious disease caused by the rickettsial organism Ehrlichia canis that affects mainly domestic dogs (Ewing, 1969; Huxsoll et al., 1970). The disease was first described in dogs by Donatien and Lestoquard (1935) in Algeria and since then it has been identified in tropical and subtropical areas all over the world (Stephenson and Ristic, 1978; Keefe et al., 1982; Brouqui et al., 1991). In Brazil, canine erhlichiosis was first diagnosed in Belo Horizonte, M.G. (Costa et al., 1973). Infected animals with CME present several clinical signs that may vary depending on the stage of the disease. Most frequent symptoms consist of high fever, anorexia, emaciation, hepatomegaly, splenomegaly, lymphadenopathy, cardiac and respiratory disturbance and nervous and ocular alterations (Walker et al., 1970; Troy and Forrester, 1990). Thrombocytopenia (Buhles et al., 1974; Davoust et al., 1991), leucopenia (Hibbler et al., 1986) and normocytic, normochromic anemia are among the major laboratory findings (Ewing and Buckner, 1965; Kuhen and Gaunt, 1985). Dogs with CME develop lesions in various organs and tissues. Pathological findings often include petechial hemorrhage in subcutaneous tissue and most organs, generalized lymphadenopathy and edema of the limbs (Hildebrandt et al., 1973). Common microscopic findings include infiltrations of lymphoreticular cells and plasma cells in many organs and tissues including nervous system, kidneys, lungs, liver and lymphoid tissues. Such cellular infiltration and lymphocyte proliferation modifies the microscopic architecture of the lymph nodes and spleen (Reardon and Pierce, 1981a,b; Valli, 1993). Generally, data on the pathogenesis of canine ehrlichiosis is scanty and vague and information is lacking about immunopathological aspects of the disease. T cell activation was proposed in the pathogenesis of the CME as was the need for the interaction of humoral and cellular immune responses for the effective intracellular destruction of the parasite (Kakoma et al., 1977; Lewis and Ristic, 1978). Also, it has been hypothesized that cellular immunity plays an important role in the pathogenesis of canine ehrlichiosis (Nyindo et al., 1980). Weiser et al. (1991) stated that an increase of T cytotoxic, killer and natural killer cell numbers probably occurs during the infection with a consequent increase in cell-mediated cytotoxicity. Page (1995) emphasized the importance of lymphocyte identification for a better understanding of most canine diseases and demonstrated a high number of CD8+ cells in peripheral blood of dogs with CME. In the present research, hematology, anti-E. canis IgG antibody titers, gross and microscopic lesions of various tissues and clinical aspects of dogs experimentally infected with E. canis were investigated. Additionally, a preliminary immunophenotyping of lymphocytes of the spleen and superficial cervical lymph nodes of dogs with CME was made in an attempt to understand alterations of lymphocyte subsets in lymphoid organs. Thus, the aim of this study was to contribute toward a better understanding of the pathogenesis of acute experimental CME.
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2. Materials and methods 2.1. Animals Eight healthy German shepherd 5–8 months, from three different litters were allocated into two groups randomized for age. Four dogs were inoculated intravenously with 5 ml each of whole blood from a dog with acute CME as described by Buhles et al. (1974). The E. canis strain was first isolated from a natural infected dog and frozen in liquid nitrogen, in the Veterinary Teaching Hospital, UNESP, Jaboticabal, SP, Brazil (unpublished data) and was inoculated in one healthy dog to obtain the inoculum (Castro et al., 1997). Four dogs served as unexposed controls. All dogs were monitored daily by physical examination. Giemsa stained blood smears from the peripheral ear vein were screened for morulae in mononuclear cells. Serum samples were obtained from all animals before the experimental inoculation and 30 days after infection to evaluate humoral responses. These samples were tested for specific IgG response to E. canis with dot-blot ELISA kit (Immunocomb® , BIOGAL). All eight dogs were seronegative to E. canis prior to exposure of the infected group. 2.2. Hematology Complete blood cell (CBC) counts and hemoglobin determination were estimated with an automated blood cell counter (CC-510 and HB-520, CELM, Barueri, SP). The packed cell volume (PCV) was determined by the microhematocrit method. Mean corpuscular volume (MCV) and mean corpuscular haemoglobin concentration (MCHC) were calculated according to Ferreira Neto et al. (1981). Blood smears stained with Giemsa were used for differential white blood cell (WBC) counts and platelet counts were performed in a Neubauer hemacytometer (Benjamin, 1979). Total plasma protein (TPP) concentration was determined using a refractometer (Jain, 1986). 2.3. Pathology All experimental dogs were euthanatized 30 days after exposure at the acute stage of the disease in the principals. Necropsy was performed and gross findings were individually recorded. Samples of spleen, lymph nodes, liver, lungs, kidneys, central nervous system (CNS), heart and intestine were collected for histopathology. Tissues were fixed in 10% phosphate-buffered formalin (pH 7.0), embedded in paraffin, cut at 5 m and stained with hematoxylin and eosin (HE). 2.4. Immunohistochemistry Immunohistochemical procedures for T cells (CD3+ and CD8+) and B cells (IgG and IgM) identification were performed on paraffin-embedded tissues or frozen sections as previously described by Caswell et al. (1995). Samples of the spleen and right superficial cervical lymph nodes were collected immediately after necropsy and fixed in phosphate-buffered 10% formalin (pH 7.0) or snap-frozen in liquid nitrogen.
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Goat anti-dog IgG (␥ chain) and IgM ( chain) were used as primary antibodies (Kirkegaard & Perry Laboratories Inc.—KPL) for B cell identification (Day, 1993). The avidin– biotin complex immunoperoxidase procedure was performed (HistomarkTM , KPL) and the slides stained with diaminobenzidine tetrahydrochloride (Diaminobenzidine reagent set, KPL). Sections were counterstained with Harris’s hematoxylin diluted 1:10 and mounted with coverslips. Histomorphometry was performed using a computerized image processing system (Videoplan, Kontron Elektronic, Carl Zeiss do Brasil). One lymph node section per animal was used to evaluate the following areas: follicular, paracortical and medulla. Folicullar region, marginal zone and splenic cords were evaluated in the spleen. Cell counts to estimate density of immunolabeled cells were performed on nine fields from each area in each lymph node and spleen sample. Mean density of cells expressing IgG or IgM in each defined region was estimated (cells × 102 /mm2 ). CD3+ lymphocytes were identified in paraffin sections according to the technique described by Day (1993) and Ferrer et al. (1992). Immunohistochemistry for CD8+ cells was performed in frozen sections of spleen and lymph nodes using anti-dog CD8 antibody (M10) (Voss et al., 1993). The follicular region and splenic cords of the spleen and follicles, and the medulla of the lymph nodes were analyzed. Histomorphometry was carried out for CD3+ and CD8+ cells as described above. Cell counts were not performed in the spleen marginal zone and lymph node paracortex due to the very high density of CD3+ and CD8+ cells that normally occur in these areas. 2.5. Statistics Data were analyzed by the analysis of variance (ANOVA) procedure and means compared by the Tukey test of SAS. Means of immunohistochemical count data were compared using Student’s t-test. 3. Results 3.1. Clinical evaluation and parasitemia detection Dogs presented clinical signs of E. canis infection between 10 and 14 days after experimental inoculation. Pale mucous membrane, lymphadenopathy, splenomegaly, hepatomegaly, emaciation and increased hair loss were observed in infected animals (Table 1). Intracytoplasmic morulae of E. canis in monocytes of blood smears were detected in all infected dogs at 12 days after infection. No clinical alterations were observed in the control group. 3.2. Hematology CBC, platelet count and TPP of infected (I) and control (C) groups are listed in Table 2. RBC, PCV and Hb values differed significantly between the two groups. Mean RBC and Hb values remained low throughout the observation period in the infected group compared to those of control animals. Significant decrease in RBC, Hb and PCV mean values of infected
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Table 1 Major clinical abnormalities observed in dogs experimentally infected with E. canisa Clinical alteration
Paleness of mucous membrane Lymphadenopathy Splenomegaly Hepatomegaly Emaciation Increased hair loss a
Animal 1
2
3
4
+ + − − + +
+ + + − + +
+ + + + + +
+ + + − + +
(+) Presence of symptoms and (−) absence of symptoms.
animals were detected at weeks 2 and 3. Infected group MCHC was significantly higher on weeks 3 and 4. WBC values in the infected group were significantly lower at weeks 2 and 3 after exposure. Eosinophil counts of the infected group declined from week 2. Infected animals showed significant increase in neutrophil and monocyte mean values at week 3 while the lymphocyte numbers decreased significantly. Platelet counts decreased significantly in infected dogs from the third week until the end of the study. TPP of infected animals decreased significantly at the third week. 3.3. Anti-E. canis IgG antibody titers All infected dogs showed positive titers for E. canis 30 days after inoculation (Table 3). 3.4. Gross and histopathological findings Necropsy of infected dogs revealed paleness of mucous membranes, subcutaneous tissues, liver and kidneys. Generalized lymphadenopathy and splenomegaly with marked hyperplasia of the white pulp were also observed in all infected dogs. Lymph nodes were edematous and the medullary area was slightly yellowish with petechial hemorrhage. Ascites and slight congestion of the lungs were observed in two animals. Histopathology of lymph nodes revealed mild folicular hyperplasia with presence of tingible body macrophages and occasional loss of discernible delimitation within the paracortical area. In addition, moderate expansion of the paracortical area and hyperplasia of the medullary cords with the presence of several plasma cells and a large number of histiocytes were observed. Moderate to severe sinus histiocytosis, erythrophagocytosis and mononuclear vasculitis were also detected. Major spleenic lesions consisted of multifocal follicular hemorrhage and congestion within the white pulp. Follicular hyperplasia, thickened splenic cords and moderate expansion of the periarteriolar lymphoid sheaths were also observed. Severe steatosis, mild to moderate perivascular and periportal mononuclear cell infiltrate and congestion of sinusoids were evident in the liver of two animals. Similar lesions were noticed in a milder degree in the two other infected dogs. Chronic glomerulonephritis and vasculitis characterized by mononuclear cell infiltrate were evident in the kidney of infected animals.
78 Table 2 Mean ± S.D. for RBC, Hb, PCV, MVC and MCHC, WBC, differential leukocyte count, platelet counting and TPP of dogs experimentally infected with E. canis (I) and control dogs (C) during experimental perioda Week 1
2
3
4
5
(×106 /l) 6.27 ± 0.52 Aa 5.36 ± 0.29 Ba
6.21 ± 0.86 Aa 4.77 ± 0.34 Bb
5.86 ± 0.61 Aa 3.23 ± 0.24 Bc
5.97 ± 0.51 Aa 2.93 ± 0.15 Bc
6.41 ± 0.43 Aa 3.25 ± 0.25 Bc
6.14 ±0.61 A 3.91 ± 1.01 B
Hb (g/dl) C I
13.89 ± 1.64 Aa 12.19 ± 1.46 Ba
13.97 ± 1.13 Aa 10.70 ± 0.79 Bb
13.86 ± 0.59 Aa 8.02 ± 0.60 Bc
14.52 ± 1.00 Aa 7.27 ± 0.54 Bc
14.62 ± 0.67 Aa 6.93 ± 0.58 Bc
14.17 ± 1.07 A 9.02 ± 2.24 B
PCV (%) C I
45.12 ± 5.69 Aa 36.12 ± 1.35 Ba
46.25 ± 4.10 Aa 33.75 ± 1.67 Ba
45.12 ± 2.29 Aa 24.87 ± 2.47 Bb
46.50 ± 3.02 Aa 21.50 ± 1.41 Bbc
45.87 ± 3.60 Aa 22.50 ± 1.77 Bc
45.77 ± 3.75 A 27.75 ± 6.32 B
MCV (fl) C I
72.01 ± 7.11 Aa 67.39 ± 1.92 Ac
75.07 ± 7.44 Aa 70.81 ± 3.19 Abc
77.43 ± 5.38 Aa 76.74 ± 3.07 Aa
78.02 ± 5.32 Aa 73.44 ± 2.89 Aab
71.54 ± 4.53 Aa 69.47 ± 6.26 Abc
74.81 ± 6.35 A 71.57 ± 4.85 B
MCHC (pg) C 30.94 ± 3.31 Aa I 33.66 ± 2.91 Aa
30.27 ± 1.48 Aa 31.68 ± 1.40 Aa
30.75 ± 1.21 Aa 32.33 ± 1.00 Ba
31.26 ± 1.54 Aa 33.85 ± 1.73 Ba
32.02 ± 2.55 Aa 30.94 ± 2.97 Aa
31.05 ± 2.14 A 32.49 ± 2.34 B
WBC (×103 /l) C 11.02 ± 1.95 Aa I 9.62 ± 2.13 Aa
10.79 ± 2.83 Aa 8.21 ± 1.63 Ba
10.37 ± 1.57 Aa 5.61 ± 1.05 Bb
10.29 ± 2.16 Aa 9.02 ± 1.86 Aa
11.45 ± 3.00 Aa 9.79 ± 1.90 Aa
10.78 ± 2.28 A 8.45 ± 2.27 B
Bands (×103 /l) C 0.18 ± 0.09 Aa I 0.20 ± 0.09 Aa
0.07 ± 0.08 Ab 0.02 ± 0.06 Ab
0.03 ± 0.05 Ab 0.09 ± 0.06 Bab
0.09 ± 0.10 Aab 0.20 ± 0.18 Ba
0.01 ± 0.03 Ab 0.23 ± 0.14 Ba
0.08 ± 0.09 A 0.16 ± 0.14 B
Neutrophils (×103 /l) C 6.52 ± 1.10 Aa I 5.61 ± 2.12 Aa
6.43 ± 1.26 Aa 5.43 ± 1.53 Aa
6.65 ± 0.94 Aa 3.79 ± 0.62 Ba
6.41 ± 1.53 Aa 5.53 ± 1.41 Aa
6.92 ± 1.26 Aa 5.77 ± 1.38 Aa
6.59 ± 1.18 A 5.23 ± 1.60 B
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RBC C I
General mean
0.57 ± 0.47 Aa 0.20 ± 0.21 Ab
0.46 ± 0.33 Aa 0 Bb
0.65 ± 0.50 Aa 0 Bb
0.70 ± 0.76 Aa 0 Bb
0.61 ± 0.52 A 0.14 ± 0.26 B
Basophils (×103 /l) C 0 I 0
0 0
0 0
0 0
0 0
0 0
Lymphocytes (×103 /l) C 3.54 ± 0.89 Aa I 3.18 ± 0.99 Aa
3.34 ± 1.74 Aa 2.02 ± 0.43 Aa
2.84 ± 0.85 Aa 0.96 ± 0.37 Bb
2.80 ± 0.92 Aa 2.18 ± 1.22 Aa
3.63 ± 1.36 Aa 3.08 ± 0.69 Aa
3.23 ± 1.20 A 2.28 ± 1.12 B
Monocytes (×103 /l) C 0.21 ± 0.23 Aa I 0.29 ± 0.19 Ab
0.35 ± 0.29 Aa 0.46 ± 0.23 Ab
0.39 ± 0.22 Aa 0.75 ± 0.42 Ba
0.40 ± 0.34 Aa 1.05 ± 0.41 Ba
0.18 ± 0.12 Aa 0.54 ± 0.28 Bb
0.31 ± 0.26 A 0.62 ± 0.41 B
Platelets (×103 /l) C 232.25 ± 24.79 Aa I 222.87 ± 35.86 Aa
237.62 ± 69.67 Aa 256.75 ± 47.89 Aa
236.00 ± 61.63 Aa 103.75 ± 22.07 Bc
228.37 ± 63.48 Aa 123.75 ± 23.48 Bbc
224.75 ± 35.19 Aa 163.12 ± 26.90 Bb
231.80 ± 2.38 A 174.00 ± 29.00 B
6.37 ± 0.63 Aa 5.64 ± 0.79 Aa
6.19 ± 0.33 Aa 5.36 ± 0.46 Ba
6.26 ± 0.26 Aa 5.06 ± 0.52 Ba
5.85 ± 0.41 Aa 5.05 ± 0.28 Ba
6.18 ± 0.08 A 5.36 ± 0.14 B
TPP (g/dl) C I
6.26 ± 0.70 Aa 5.72 ± 0.94 Aa
a Different capital letters in a column and different lower case letters on a line indicate statistical significant differences (P < 0.05—Tukey test) between groups and weeks, respectively.
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Eosinophils (×103 /l) C 0.66 ± 0.55 Aa I 0.50 ± 0.33 Aa
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Table 3 Titers of IgG antibodies (Immunocomb® ) to E. canis in dogs experimentally infected with E. canis at 30 days after infection Animal
Colorimetric scalea
Serological titers
1 2 3 4
3 2 4 2
1:160 1:80 1:320 1:80
a
Colorimetric scale for acquire correspondent serological titer according.
Mononuclear cell meningitis and presence of broad perivascular cuffs of mononuclear cells affecting the cerebral cortex, cerebellum and medulla were observed. Small foci of mononuclear cell infiltration in the alveolar septum and mononuclear cell vasculitis were evident in the lungs of all infected animals. No gross or microscopic alterations were observed in control animals. 3.5. Immunohistochemistry The mean values of immunolabeled cells of the lymph nodes and spleen of infected and control dogs are listed in Table 4.
Plate 1. Immunohistochemistry of lymph nodes of dogs experimentally infected with E. canis. (A) Positive IgG cells in follicular area; (B) positive IgM cells in follicular area; (C) positive CD3 cells in follicular areas; (D) positive CD8 cells in follicular areas (original magnification: 200×).
Organ
Lymph node Lymph node Lymph node Spleen Spleen Spleen ∗
Region
Follicular Paracortex Medulla Follicular Marginal zone Splenic cords
P > 0.05 (Student’s t-test).
IgG (cells × 102 /mm2 )
IgM (cells × 102 /mm2 )
CD3 (cells × 102 /mm2 )
CD8 (cells × 102 /mm2 )
C
I
C
I
C
I
C
I
20.52 ± 9.61 12.01 ± 6.37 21.47 ± 4.55 2.79 ± 1.43 6.94 ± 2.68 4.08 ± 1.69
5.71 ± 3.80 56.12 ± 14.58 68.87 ± 13.14 5.10 ± 2.00 58.51 ± 19.54 42.94 ± 8.80
7.83 ± 7.17 5.46 ± 4.87 5.82 ± 3.56 2.03 ± 1.39∗ 4.70 ± 2.55 1.75 ± 1.33
1.78 ± 1.19 13.70 ± 5.16 9.77 ± 2.90 2.43 ± 1.51∗ 18.61 ± 7.20 6.30 ± 3.11
14.50 ± 4.61 – 14.31 ± 4.63 9.58 ± 5.18 – 19.04 ± 3.80
4.27 ± 2.81 – 46.90 ± 12.63 17.81 ± 5.92 – 35.94 ± 6.01
1.04 ± 0.87 – 16.59 ± 2.94 7.68 ± 2.13∗ – 27.15 ± 5.95
11.12 ± 5.29 – 37.94 ± 5.18 5.34 ± 3.88∗ – 17.75 ± 7.01
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Table 4 Distribution of IgG, IgM, CD3 and CD8 labeled cells (mean ± S.D.) analyzed in different regions of lymph nodes and spleen of principal (I) and control dogs (C)
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The number of immunolabeled IgG cells (Plate 1) in paracortical and medullary regions of lymph nodes and also in splenic cords and marginal zone of the spleen was higher in infected dogs. Immunolabeled IgM cells were present in lower numbers in the lymph nodes (Plate 1) of control animals than in infected animals, except for the follicular region. Similarly, IgM cells were found in higher numbers in the spleen of infected animals. Infected animals showed a higher number of CD3+ cells in the medulla of lymph nodes and in the spleen. In contrast, CD3+ cells predominated in follicular regions of control animal lymph nodes (Plate 1). Inoculated dogs had a higher number of CD8+ cells in medullary and follicular regions of lymph nodes (Plate 1) and a lower number in splenic cords than did control dogs.
4. Discussion Pathogenesis of CME still remains unknown. Immunologic dysfunction has been suggested as a mechanism for pathologic alterations and clinical signs. Fever and some clinical signs and symptoms observed in all infected dogs may have been caused by increased production of interleukin-1 (IL-1) by antigen presenting cells and B cells or exogenous pyrogen products of the parasite (Gershwin et al., 1995). These alterations were already evident between days 10 and 14 after infection, similar to the findings reported in naturally infected and dogs experimentally exposed to E. canis by Huxsoll et al. (1972) and Pierce et al. (1977), respectively. Other clinical findings such as pale mucous membranes, lymphadenopathy, splenomegaly, hepatomegaly, emaciation, increased hair loss and hematological alterations observed in these infected animals have been previously reported in cases of canine ehrlichiosis (Hoskins, 1991; Troy and Forrester, 1990). Anemia, mild thrombocytopenia and leucopenia were observed and are very frequently related to CME (Troy and Forrester, 1990; Davoust et al., 1991). Infected dogs presented mild to moderate hematological changes in acute experimental infection for just a few weeks. The tendency for the hematological parameters to return to normal was evident at the end of the experiment. This result may be a consequence of transient suppression of bone marrow activity due to E. canis infection (Buhles et al., 1974, 1975). Anti-platelet antibodies have been demonstrated in CME and have been blamed for the pathogenesis of thrombocytopenia (Harrus et al., 1996). Anemia may explain the observed paleness of mucous membranes and most organs. Lymphadenopathy, splenomegaly, ascites, paleness of mucous membrane, kidney and liver and discrete pulmonary congestion were observed at the necropsy of inoculated dogs as has been previously reported in cases of canine ehrlichiosis (Hildebrandt et al., 1970, 1973). Microscopic examination of the lymph nodes revealed increased lymphocyte numbers in medullary and paracortical regions and might explain the lymphadenopathy observed in all infected dogs of this study. Nyindo et al. (1980) observed the same in German Shepherd dogs and posited breed susceptibility. Medullary cord hyperplasia and increased number of histiocytes and plasma cells in the lymph nodes, as well as white pulp and splenic cord hyperplasia in the spleen are a consequence of the immune response. These lesions
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are similar to those reported by Reardon and Pierce (1981a) who demonstrated later that these lymphoid changes did not occur in experimentally immunosuppressed animals, incriminating the immune system in development of lesions (Reardon and Pierce, 1981b). Harrus et al. (1998) demonstrated that the spleen plays an important role in the pathogenesis of CME and proposed that splenic substances and/or inflammatory mediators are involved. Ascites in two animals could have resulted from vasculitis or hypoalbminemia associated with hypergamaglobulinemia since the infected dogs showed total protein levels within normal ranges (Van Dijk, 1971; Simpson, 1974; Hoskins, 1991). Hepatic, renal and CNS lesions may have occurred due to compressive circulatory and/or immunomediated alterations. Hepatic lesions have been reported by Reardon and Pierce (1981a) in dogs with canine ehrlichiosis. Vasculitis was a consistent finding in tissues of all infected animals. It is possible that the general inflammation observed in the vessels (vasculitis) contributes to the pathogenesis of the disease (Van Dijk, 1971; Simpson, 1974). Immunohistochemistry of lymphoid organs revealed increased number of IgM and IgG immunolabeled cells in dogs with CME. Such findings associated with an increase in specific anti-E. canis IgG antibody titers, indicate that humoral responses may play an important role in CME (Cadman et al., 1994). IgG and IgM immunolabeled cell populations are, at least in part, responsible for the morphological changes observed in the spleen and lymph nodes. This observation was also suggested by Reardon and Pierce (1981a), even though these authors did not perform immunohistochemical analysis. In humoral immune response to E. canis IgG2 is predominant in natural and experimental infection (Harrus et al., 2001). Anti-E. canis-IgG2 serum levels were found to be higher than anti-E. canis-IgG1 during the different phases of the disease in symptomatic and asymptomatic dogs (Harrus et al., 2001). IgG immunoglobulins are known to activate complement and thus participate in type III hypersensitivity reactions (Abbas et al., 2000). It is tempting to speculate that such mechanism have a role in the vasculitis observed in CME. CD3+ cells were present in higher numbers in the splenic cords and in the medullary region of lymph nodes, and in lower numbers in the follicular region of the lymph nodes of all infected dogs when compared to control animals. The lower number of CD3+ cells in the follicular region of the lymph nodes may represent an important finding. CD3+ cells found in follicular areas are probably T helper CD4+ cells (Caswell et al., 1995). Based on this observation, a decreased number of T helper cells may be indicative of immunological dysfunction in dogs with CME. Unfortunately, CD4+ T cells could not be immunophenotyped by the time of the conduct of this work by the lack of specific immune reagents in our laboratory. In this regard, an additional issue to be studied in future experimental projects of CME pathogenesis would be the cytokine profile produced by CD4+ cells during infection. It is already well-established that different subsets of CD4+ cells (Th1 or Th2 ) may be responsible for very different outcome in infectious diseases (Mosmann and Sad, 1996; Meusen, 1998). CD8+ cell populations were increased in the follicular and medullary regions of the lymph nodes of animals infected with E. canis. This finding demonstrates that cell-mediated responses were pronounced in inoculated dogs. Kakoma et al. (1977) and Nyindo et al. (1980) reported the importance of cellular immune response in the clinical development
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of the disease. Our finding supports observations made by Page (1995) who reported an increased number of CD8+ cells in peripheral blood of dogs with canine ehrlichiosis. On the other hand, CD8+ cells were decreased in the spleen of infected dogs which could have occurred as an immune response imbalance in this organ in CME. CD8+ cells may represent a protective immune response against persistent E. canis that resists eradication by phagocytes and antibodies while it can also mediate tissue injury by cytolytic elimination of parasitized host cells and thus contribute to CME pathogenesis. Broadly, the present study shows an intense involvement of immune cells in canine ehrlichiosis, especially in lymph nodes and spleen. Immune-mediated vasculitis may play a central role in the pathogenesis of CME and could explain most of the important lesions observed in organs and tissues of infected dogs. Severity of lesions in infected animals in the presence of an intense immune activity suggests immune-mediated mechanisms in the genesis of lesions. Additional studies are being conducted in an attempt to elucidate the mechanisms involved in cell-mediated immune responses in CME.
References ˙ Abbas, A.K., Lichtman, A.H., Pober, J.S. (Eds.), 2000. Cellular and Mollecular Immunology. W.B. Saunders Company, 553 pp. Benjamin, M.M., 1979. Counting of blood cells. In: Outline of Veterinary Clinical Pathology, third ed. The Iowa State University Press, Iowa, pp. 48–59. Brouqui, P., Davoust, B., Haddad, S., Vidor, E., Raoult, D., 1991. Serological evaluation of Erlichia canis infection in military dogs in Africa and Reunion Island. Vet. Microbiol. 26, 103–105. Buhles Jr., W.C., Huxsoll, D.L., Ristic, M., 1974. Tropical Canine Thrombocytopenia: clinical, hematologic, and serologic response of dogs to Erlichia canis infection, tetracycline therapy, and challenge inoculation. J. Infect. Dis. 130, 357–367. Buhles Jr., W.C., Huxsoll, D.L., Hildebrandt, P.K., 1975. Tropical Canine Pancitopenia: role of aplastic anemia in the pathogenesis of severe disease. J. Comp. Pathol. 85, 511–521. Cadman, H.F., Kelly, P.J., Matthewman, L.A., Zhou, R., Mason, P.R., 1994. Comparison of the dot-blot enzyme linked immunoassay with immunofluorescence for detecting antibodies to Ehrlichia canis. Vet. Rec. 135, 362. Castro, M.B., Machado, R.Z., Alessi, A.C., Aquino, L.P.C.T., 1997. Ehrlichia canis (Donatien and Lestoquard, 1935): experimental infection in dogs. In: Proceedings of the IVth Biennial Meeting Society for Tropical Veterinary Medicine, Montpellier, French. Caswell, J.L., Yager, J.A., Ferrer, L., Weir, J.A.M., 1995. Canine demodicosis: a re-examination of the histopatologic lesions and description of the immunophenotype of infiltrating cells. Vet. Dermatol. 6, 9–19. Costa, J.O., Batista Júnior, J.A., Silva, M., Guimarães, P.M., 1973. Ehrlichia canis infection in dog in Belo Horizonte, Brazil. Arq. Esc. Vet. UFMG 25, 199–200. Davoust, B., Parzy, D., Vidor, E., Hasselot, N., Martet, G., 1991. Ehrlichiose Canine Experimentale: étude clinique et therapeutique. Réc. Méd. Vet. 167, 33–40. Day, M.J., 1993. Immunopathology of anal furunculosis in the dog. J. Small Anim. Pract. 34, 381–389. Donatien, A., Lestoquard, F., 1935. Existence en Algére d’une Rickettsia du chien. Bull. Soc. Pathol. Exot. 28, 418–419. Ewing, S.A., 1969. Canine Ehrlichiosis. Adv. Vet. Sci. Comp. Med. 13, 331–353. Ewing, S.A., Buckner, R.G., 1965. Manifestation of babesiosis, ehrlichiosis and combined infections in the dog. Am. J. Vet. Res. 26, 815–828. Ferrer, L., Fondevila, D., Rabanal, R., Ramis, A., 1992. Detection of T lymphocytes in canine tissue embedded in paraffin wax by means of antibody to CD3 antigen. J. Comp. Pathol. 106, 311–314. Ferreira Neto, J.M., Viana, E.S., Magalhães, L.M. (Eds.) 1981. Patologia Cl´ınica Veterinária. Editora Rabelo, Belo Horizonte, 279 p.
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Experimental acute canine monocytic ehrlichiosis: clinicopathological and immunopathological findings Márcio Botelho de Castro a,b , Rosangela Zacarias Machado b,∗ , Lucia Padilha Cury Tomaz de Aquino a,b , Antonio Carlos Alessi b , Mirela Tinucci Costa c a
Universidade de Franca, Av. Dr. Armando Salles de Oliveira, 201, CEP 14404-600 Franca, SP, Brazil b Department of Veterinary Pathology, Universidade Estadual Paulista (UNESP), Via de Acesso Prof. Paulo Donato Castellani, sem no. CEP 14870-000 Jaboticabal, SP, Brazil c Veterinary Teaching Hospital, Universidade Estadual Paulista (UNESP), Via de Acesso Prof. Paulo Donato Castellani, sem no., CEP 14870-000 Jaboticabal, SP, Brazil Received 1 September 2002; received in revised form 25 September 2003; accepted 16 October 2003
Abstract Clinical signs, humoral and cellular immune responses, and microscopic and gross tissue alterations resulting from acute experimental Ehrlichia canis infection in dogs were studied. Four dogs were inoculated with E. canis and four were used as uninfected controls. After a 10–14-day incubation period, infected dogs developed pyrexia up to 41 ◦ C for 6–8 days. Antibody titers to E. canis antigen were demonstrable in all inoculated dogs at 30 days post-infection. Necropsy of infected animals revealed pale mucous membranes, generalized lymphadenopathy, splenomegaly, edema and ascites. Microcopically, the main lesions were: lymphoreticular hyperplasia in cortical areas of lymph nodes and spleenic white pulp, periportal accumulation of mononuclear cells and centrolobular fatty degeneration of the liver. Kidneys presented with glomerulonephritis characterized by interstitial mononuclear infiltration. Immunophenotyping of lymphocytes from lymph nodes and spleen sections displayed alterations in IgG, IgM, CD3+ and CD8+ cells population in infected dogs. © 2003 Elsevier B.V. All rights reserved. Keywords: Ehrlichia canis; Ehrlichiosis; Dog; Immunopathology; Immunohistochemistry
∗ Corresponding author. Tel.: +55-16-32092664; fax: +55-16-32024275. E-mail address: [email protected] (R.Z. Machado).
0304-4017/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2003.10.012
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1. Introduction Canine monocytic ehrlichiosis (CME) is an infectious disease caused by the rickettsial organism Ehrlichia canis that affects mainly domestic dogs (Ewing, 1969; Huxsoll et al., 1970). The disease was first described in dogs by Donatien and Lestoquard (1935) in Algeria and since then it has been identified in tropical and subtropical areas all over the world (Stephenson and Ristic, 1978; Keefe et al., 1982; Brouqui et al., 1991). In Brazil, canine erhlichiosis was first diagnosed in Belo Horizonte, M.G. (Costa et al., 1973). Infected animals with CME present several clinical signs that may vary depending on the stage of the disease. Most frequent symptoms consist of high fever, anorexia, emaciation, hepatomegaly, splenomegaly, lymphadenopathy, cardiac and respiratory disturbance and nervous and ocular alterations (Walker et al., 1970; Troy and Forrester, 1990). Thrombocytopenia (Buhles et al., 1974; Davoust et al., 1991), leucopenia (Hibbler et al., 1986) and normocytic, normochromic anemia are among the major laboratory findings (Ewing and Buckner, 1965; Kuhen and Gaunt, 1985). Dogs with CME develop lesions in various organs and tissues. Pathological findings often include petechial hemorrhage in subcutaneous tissue and most organs, generalized lymphadenopathy and edema of the limbs (Hildebrandt et al., 1973). Common microscopic findings include infiltrations of lymphoreticular cells and plasma cells in many organs and tissues including nervous system, kidneys, lungs, liver and lymphoid tissues. Such cellular infiltration and lymphocyte proliferation modifies the microscopic architecture of the lymph nodes and spleen (Reardon and Pierce, 1981a,b; Valli, 1993). Generally, data on the pathogenesis of canine ehrlichiosis is scanty and vague and information is lacking about immunopathological aspects of the disease. T cell activation was proposed in the pathogenesis of the CME as was the need for the interaction of humoral and cellular immune responses for the effective intracellular destruction of the parasite (Kakoma et al., 1977; Lewis and Ristic, 1978). Also, it has been hypothesized that cellular immunity plays an important role in the pathogenesis of canine ehrlichiosis (Nyindo et al., 1980). Weiser et al. (1991) stated that an increase of T cytotoxic, killer and natural killer cell numbers probably occurs during the infection with a consequent increase in cell-mediated cytotoxicity. Page (1995) emphasized the importance of lymphocyte identification for a better understanding of most canine diseases and demonstrated a high number of CD8+ cells in peripheral blood of dogs with CME. In the present research, hematology, anti-E. canis IgG antibody titers, gross and microscopic lesions of various tissues and clinical aspects of dogs experimentally infected with E. canis were investigated. Additionally, a preliminary immunophenotyping of lymphocytes of the spleen and superficial cervical lymph nodes of dogs with CME was made in an attempt to understand alterations of lymphocyte subsets in lymphoid organs. Thus, the aim of this study was to contribute toward a better understanding of the pathogenesis of acute experimental CME.
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2. Materials and methods 2.1. Animals Eight healthy German shepherd 5–8 months, from three different litters were allocated into two groups randomized for age. Four dogs were inoculated intravenously with 5 ml each of whole blood from a dog with acute CME as described by Buhles et al. (1974). The E. canis strain was first isolated from a natural infected dog and frozen in liquid nitrogen, in the Veterinary Teaching Hospital, UNESP, Jaboticabal, SP, Brazil (unpublished data) and was inoculated in one healthy dog to obtain the inoculum (Castro et al., 1997). Four dogs served as unexposed controls. All dogs were monitored daily by physical examination. Giemsa stained blood smears from the peripheral ear vein were screened for morulae in mononuclear cells. Serum samples were obtained from all animals before the experimental inoculation and 30 days after infection to evaluate humoral responses. These samples were tested for specific IgG response to E. canis with dot-blot ELISA kit (Immunocomb® , BIOGAL). All eight dogs were seronegative to E. canis prior to exposure of the infected group. 2.2. Hematology Complete blood cell (CBC) counts and hemoglobin determination were estimated with an automated blood cell counter (CC-510 and HB-520, CELM, Barueri, SP). The packed cell volume (PCV) was determined by the microhematocrit method. Mean corpuscular volume (MCV) and mean corpuscular haemoglobin concentration (MCHC) were calculated according to Ferreira Neto et al. (1981). Blood smears stained with Giemsa were used for differential white blood cell (WBC) counts and platelet counts were performed in a Neubauer hemacytometer (Benjamin, 1979). Total plasma protein (TPP) concentration was determined using a refractometer (Jain, 1986). 2.3. Pathology All experimental dogs were euthanatized 30 days after exposure at the acute stage of the disease in the principals. Necropsy was performed and gross findings were individually recorded. Samples of spleen, lymph nodes, liver, lungs, kidneys, central nervous system (CNS), heart and intestine were collected for histopathology. Tissues were fixed in 10% phosphate-buffered formalin (pH 7.0), embedded in paraffin, cut at 5 m and stained with hematoxylin and eosin (HE). 2.4. Immunohistochemistry Immunohistochemical procedures for T cells (CD3+ and CD8+) and B cells (IgG and IgM) identification were performed on paraffin-embedded tissues or frozen sections as previously described by Caswell et al. (1995). Samples of the spleen and right superficial cervical lymph nodes were collected immediately after necropsy and fixed in phosphate-buffered 10% formalin (pH 7.0) or snap-frozen in liquid nitrogen.
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Goat anti-dog IgG (␥ chain) and IgM ( chain) were used as primary antibodies (Kirkegaard & Perry Laboratories Inc.—KPL) for B cell identification (Day, 1993). The avidin– biotin complex immunoperoxidase procedure was performed (HistomarkTM , KPL) and the slides stained with diaminobenzidine tetrahydrochloride (Diaminobenzidine reagent set, KPL). Sections were counterstained with Harris’s hematoxylin diluted 1:10 and mounted with coverslips. Histomorphometry was performed using a computerized image processing system (Videoplan, Kontron Elektronic, Carl Zeiss do Brasil). One lymph node section per animal was used to evaluate the following areas: follicular, paracortical and medulla. Folicullar region, marginal zone and splenic cords were evaluated in the spleen. Cell counts to estimate density of immunolabeled cells were performed on nine fields from each area in each lymph node and spleen sample. Mean density of cells expressing IgG or IgM in each defined region was estimated (cells × 102 /mm2 ). CD3+ lymphocytes were identified in paraffin sections according to the technique described by Day (1993) and Ferrer et al. (1992). Immunohistochemistry for CD8+ cells was performed in frozen sections of spleen and lymph nodes using anti-dog CD8 antibody (M10) (Voss et al., 1993). The follicular region and splenic cords of the spleen and follicles, and the medulla of the lymph nodes were analyzed. Histomorphometry was carried out for CD3+ and CD8+ cells as described above. Cell counts were not performed in the spleen marginal zone and lymph node paracortex due to the very high density of CD3+ and CD8+ cells that normally occur in these areas. 2.5. Statistics Data were analyzed by the analysis of variance (ANOVA) procedure and means compared by the Tukey test of SAS. Means of immunohistochemical count data were compared using Student’s t-test. 3. Results 3.1. Clinical evaluation and parasitemia detection Dogs presented clinical signs of E. canis infection between 10 and 14 days after experimental inoculation. Pale mucous membrane, lymphadenopathy, splenomegaly, hepatomegaly, emaciation and increased hair loss were observed in infected animals (Table 1). Intracytoplasmic morulae of E. canis in monocytes of blood smears were detected in all infected dogs at 12 days after infection. No clinical alterations were observed in the control group. 3.2. Hematology CBC, platelet count and TPP of infected (I) and control (C) groups are listed in Table 2. RBC, PCV and Hb values differed significantly between the two groups. Mean RBC and Hb values remained low throughout the observation period in the infected group compared to those of control animals. Significant decrease in RBC, Hb and PCV mean values of infected
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Table 1 Major clinical abnormalities observed in dogs experimentally infected with E. canisa Clinical alteration
Paleness of mucous membrane Lymphadenopathy Splenomegaly Hepatomegaly Emaciation Increased hair loss a
Animal 1
2
3
4
+ + − − + +
+ + + − + +
+ + + + + +
+ + + − + +
(+) Presence of symptoms and (−) absence of symptoms.
animals were detected at weeks 2 and 3. Infected group MCHC was significantly higher on weeks 3 and 4. WBC values in the infected group were significantly lower at weeks 2 and 3 after exposure. Eosinophil counts of the infected group declined from week 2. Infected animals showed significant increase in neutrophil and monocyte mean values at week 3 while the lymphocyte numbers decreased significantly. Platelet counts decreased significantly in infected dogs from the third week until the end of the study. TPP of infected animals decreased significantly at the third week. 3.3. Anti-E. canis IgG antibody titers All infected dogs showed positive titers for E. canis 30 days after inoculation (Table 3). 3.4. Gross and histopathological findings Necropsy of infected dogs revealed paleness of mucous membranes, subcutaneous tissues, liver and kidneys. Generalized lymphadenopathy and splenomegaly with marked hyperplasia of the white pulp were also observed in all infected dogs. Lymph nodes were edematous and the medullary area was slightly yellowish with petechial hemorrhage. Ascites and slight congestion of the lungs were observed in two animals. Histopathology of lymph nodes revealed mild folicular hyperplasia with presence of tingible body macrophages and occasional loss of discernible delimitation within the paracortical area. In addition, moderate expansion of the paracortical area and hyperplasia of the medullary cords with the presence of several plasma cells and a large number of histiocytes were observed. Moderate to severe sinus histiocytosis, erythrophagocytosis and mononuclear vasculitis were also detected. Major spleenic lesions consisted of multifocal follicular hemorrhage and congestion within the white pulp. Follicular hyperplasia, thickened splenic cords and moderate expansion of the periarteriolar lymphoid sheaths were also observed. Severe steatosis, mild to moderate perivascular and periportal mononuclear cell infiltrate and congestion of sinusoids were evident in the liver of two animals. Similar lesions were noticed in a milder degree in the two other infected dogs. Chronic glomerulonephritis and vasculitis characterized by mononuclear cell infiltrate were evident in the kidney of infected animals.
78 Table 2 Mean ± S.D. for RBC, Hb, PCV, MVC and MCHC, WBC, differential leukocyte count, platelet counting and TPP of dogs experimentally infected with E. canis (I) and control dogs (C) during experimental perioda Week 1
2
3
4
5
(×106 /l) 6.27 ± 0.52 Aa 5.36 ± 0.29 Ba
6.21 ± 0.86 Aa 4.77 ± 0.34 Bb
5.86 ± 0.61 Aa 3.23 ± 0.24 Bc
5.97 ± 0.51 Aa 2.93 ± 0.15 Bc
6.41 ± 0.43 Aa 3.25 ± 0.25 Bc
6.14 ±0.61 A 3.91 ± 1.01 B
Hb (g/dl) C I
13.89 ± 1.64 Aa 12.19 ± 1.46 Ba
13.97 ± 1.13 Aa 10.70 ± 0.79 Bb
13.86 ± 0.59 Aa 8.02 ± 0.60 Bc
14.52 ± 1.00 Aa 7.27 ± 0.54 Bc
14.62 ± 0.67 Aa 6.93 ± 0.58 Bc
14.17 ± 1.07 A 9.02 ± 2.24 B
PCV (%) C I
45.12 ± 5.69 Aa 36.12 ± 1.35 Ba
46.25 ± 4.10 Aa 33.75 ± 1.67 Ba
45.12 ± 2.29 Aa 24.87 ± 2.47 Bb
46.50 ± 3.02 Aa 21.50 ± 1.41 Bbc
45.87 ± 3.60 Aa 22.50 ± 1.77 Bc
45.77 ± 3.75 A 27.75 ± 6.32 B
MCV (fl) C I
72.01 ± 7.11 Aa 67.39 ± 1.92 Ac
75.07 ± 7.44 Aa 70.81 ± 3.19 Abc
77.43 ± 5.38 Aa 76.74 ± 3.07 Aa
78.02 ± 5.32 Aa 73.44 ± 2.89 Aab
71.54 ± 4.53 Aa 69.47 ± 6.26 Abc
74.81 ± 6.35 A 71.57 ± 4.85 B
MCHC (pg) C 30.94 ± 3.31 Aa I 33.66 ± 2.91 Aa
30.27 ± 1.48 Aa 31.68 ± 1.40 Aa
30.75 ± 1.21 Aa 32.33 ± 1.00 Ba
31.26 ± 1.54 Aa 33.85 ± 1.73 Ba
32.02 ± 2.55 Aa 30.94 ± 2.97 Aa
31.05 ± 2.14 A 32.49 ± 2.34 B
WBC (×103 /l) C 11.02 ± 1.95 Aa I 9.62 ± 2.13 Aa
10.79 ± 2.83 Aa 8.21 ± 1.63 Ba
10.37 ± 1.57 Aa 5.61 ± 1.05 Bb
10.29 ± 2.16 Aa 9.02 ± 1.86 Aa
11.45 ± 3.00 Aa 9.79 ± 1.90 Aa
10.78 ± 2.28 A 8.45 ± 2.27 B
Bands (×103 /l) C 0.18 ± 0.09 Aa I 0.20 ± 0.09 Aa
0.07 ± 0.08 Ab 0.02 ± 0.06 Ab
0.03 ± 0.05 Ab 0.09 ± 0.06 Bab
0.09 ± 0.10 Aab 0.20 ± 0.18 Ba
0.01 ± 0.03 Ab 0.23 ± 0.14 Ba
0.08 ± 0.09 A 0.16 ± 0.14 B
Neutrophils (×103 /l) C 6.52 ± 1.10 Aa I 5.61 ± 2.12 Aa
6.43 ± 1.26 Aa 5.43 ± 1.53 Aa
6.65 ± 0.94 Aa 3.79 ± 0.62 Ba
6.41 ± 1.53 Aa 5.53 ± 1.41 Aa
6.92 ± 1.26 Aa 5.77 ± 1.38 Aa
6.59 ± 1.18 A 5.23 ± 1.60 B
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RBC C I
General mean
0.57 ± 0.47 Aa 0.20 ± 0.21 Ab
0.46 ± 0.33 Aa 0 Bb
0.65 ± 0.50 Aa 0 Bb
0.70 ± 0.76 Aa 0 Bb
0.61 ± 0.52 A 0.14 ± 0.26 B
Basophils (×103 /l) C 0 I 0
0 0
0 0
0 0
0 0
0 0
Lymphocytes (×103 /l) C 3.54 ± 0.89 Aa I 3.18 ± 0.99 Aa
3.34 ± 1.74 Aa 2.02 ± 0.43 Aa
2.84 ± 0.85 Aa 0.96 ± 0.37 Bb
2.80 ± 0.92 Aa 2.18 ± 1.22 Aa
3.63 ± 1.36 Aa 3.08 ± 0.69 Aa
3.23 ± 1.20 A 2.28 ± 1.12 B
Monocytes (×103 /l) C 0.21 ± 0.23 Aa I 0.29 ± 0.19 Ab
0.35 ± 0.29 Aa 0.46 ± 0.23 Ab
0.39 ± 0.22 Aa 0.75 ± 0.42 Ba
0.40 ± 0.34 Aa 1.05 ± 0.41 Ba
0.18 ± 0.12 Aa 0.54 ± 0.28 Bb
0.31 ± 0.26 A 0.62 ± 0.41 B
Platelets (×103 /l) C 232.25 ± 24.79 Aa I 222.87 ± 35.86 Aa
237.62 ± 69.67 Aa 256.75 ± 47.89 Aa
236.00 ± 61.63 Aa 103.75 ± 22.07 Bc
228.37 ± 63.48 Aa 123.75 ± 23.48 Bbc
224.75 ± 35.19 Aa 163.12 ± 26.90 Bb
231.80 ± 2.38 A 174.00 ± 29.00 B
6.37 ± 0.63 Aa 5.64 ± 0.79 Aa
6.19 ± 0.33 Aa 5.36 ± 0.46 Ba
6.26 ± 0.26 Aa 5.06 ± 0.52 Ba
5.85 ± 0.41 Aa 5.05 ± 0.28 Ba
6.18 ± 0.08 A 5.36 ± 0.14 B
TPP (g/dl) C I
6.26 ± 0.70 Aa 5.72 ± 0.94 Aa
a Different capital letters in a column and different lower case letters on a line indicate statistical significant differences (P < 0.05—Tukey test) between groups and weeks, respectively.
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Eosinophils (×103 /l) C 0.66 ± 0.55 Aa I 0.50 ± 0.33 Aa
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Table 3 Titers of IgG antibodies (Immunocomb® ) to E. canis in dogs experimentally infected with E. canis at 30 days after infection Animal
Colorimetric scalea
Serological titers
1 2 3 4
3 2 4 2
1:160 1:80 1:320 1:80
a
Colorimetric scale for acquire correspondent serological titer according.
Mononuclear cell meningitis and presence of broad perivascular cuffs of mononuclear cells affecting the cerebral cortex, cerebellum and medulla were observed. Small foci of mononuclear cell infiltration in the alveolar septum and mononuclear cell vasculitis were evident in the lungs of all infected animals. No gross or microscopic alterations were observed in control animals. 3.5. Immunohistochemistry The mean values of immunolabeled cells of the lymph nodes and spleen of infected and control dogs are listed in Table 4.
Plate 1. Immunohistochemistry of lymph nodes of dogs experimentally infected with E. canis. (A) Positive IgG cells in follicular area; (B) positive IgM cells in follicular area; (C) positive CD3 cells in follicular areas; (D) positive CD8 cells in follicular areas (original magnification: 200×).
Organ
Lymph node Lymph node Lymph node Spleen Spleen Spleen ∗
Region
Follicular Paracortex Medulla Follicular Marginal zone Splenic cords
P > 0.05 (Student’s t-test).
IgG (cells × 102 /mm2 )
IgM (cells × 102 /mm2 )
CD3 (cells × 102 /mm2 )
CD8 (cells × 102 /mm2 )
C
I
C
I
C
I
C
I
20.52 ± 9.61 12.01 ± 6.37 21.47 ± 4.55 2.79 ± 1.43 6.94 ± 2.68 4.08 ± 1.69
5.71 ± 3.80 56.12 ± 14.58 68.87 ± 13.14 5.10 ± 2.00 58.51 ± 19.54 42.94 ± 8.80
7.83 ± 7.17 5.46 ± 4.87 5.82 ± 3.56 2.03 ± 1.39∗ 4.70 ± 2.55 1.75 ± 1.33
1.78 ± 1.19 13.70 ± 5.16 9.77 ± 2.90 2.43 ± 1.51∗ 18.61 ± 7.20 6.30 ± 3.11
14.50 ± 4.61 – 14.31 ± 4.63 9.58 ± 5.18 – 19.04 ± 3.80
4.27 ± 2.81 – 46.90 ± 12.63 17.81 ± 5.92 – 35.94 ± 6.01
1.04 ± 0.87 – 16.59 ± 2.94 7.68 ± 2.13∗ – 27.15 ± 5.95
11.12 ± 5.29 – 37.94 ± 5.18 5.34 ± 3.88∗ – 17.75 ± 7.01
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Table 4 Distribution of IgG, IgM, CD3 and CD8 labeled cells (mean ± S.D.) analyzed in different regions of lymph nodes and spleen of principal (I) and control dogs (C)
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The number of immunolabeled IgG cells (Plate 1) in paracortical and medullary regions of lymph nodes and also in splenic cords and marginal zone of the spleen was higher in infected dogs. Immunolabeled IgM cells were present in lower numbers in the lymph nodes (Plate 1) of control animals than in infected animals, except for the follicular region. Similarly, IgM cells were found in higher numbers in the spleen of infected animals. Infected animals showed a higher number of CD3+ cells in the medulla of lymph nodes and in the spleen. In contrast, CD3+ cells predominated in follicular regions of control animal lymph nodes (Plate 1). Inoculated dogs had a higher number of CD8+ cells in medullary and follicular regions of lymph nodes (Plate 1) and a lower number in splenic cords than did control dogs.
4. Discussion Pathogenesis of CME still remains unknown. Immunologic dysfunction has been suggested as a mechanism for pathologic alterations and clinical signs. Fever and some clinical signs and symptoms observed in all infected dogs may have been caused by increased production of interleukin-1 (IL-1) by antigen presenting cells and B cells or exogenous pyrogen products of the parasite (Gershwin et al., 1995). These alterations were already evident between days 10 and 14 after infection, similar to the findings reported in naturally infected and dogs experimentally exposed to E. canis by Huxsoll et al. (1972) and Pierce et al. (1977), respectively. Other clinical findings such as pale mucous membranes, lymphadenopathy, splenomegaly, hepatomegaly, emaciation, increased hair loss and hematological alterations observed in these infected animals have been previously reported in cases of canine ehrlichiosis (Hoskins, 1991; Troy and Forrester, 1990). Anemia, mild thrombocytopenia and leucopenia were observed and are very frequently related to CME (Troy and Forrester, 1990; Davoust et al., 1991). Infected dogs presented mild to moderate hematological changes in acute experimental infection for just a few weeks. The tendency for the hematological parameters to return to normal was evident at the end of the experiment. This result may be a consequence of transient suppression of bone marrow activity due to E. canis infection (Buhles et al., 1974, 1975). Anti-platelet antibodies have been demonstrated in CME and have been blamed for the pathogenesis of thrombocytopenia (Harrus et al., 1996). Anemia may explain the observed paleness of mucous membranes and most organs. Lymphadenopathy, splenomegaly, ascites, paleness of mucous membrane, kidney and liver and discrete pulmonary congestion were observed at the necropsy of inoculated dogs as has been previously reported in cases of canine ehrlichiosis (Hildebrandt et al., 1970, 1973). Microscopic examination of the lymph nodes revealed increased lymphocyte numbers in medullary and paracortical regions and might explain the lymphadenopathy observed in all infected dogs of this study. Nyindo et al. (1980) observed the same in German Shepherd dogs and posited breed susceptibility. Medullary cord hyperplasia and increased number of histiocytes and plasma cells in the lymph nodes, as well as white pulp and splenic cord hyperplasia in the spleen are a consequence of the immune response. These lesions
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are similar to those reported by Reardon and Pierce (1981a) who demonstrated later that these lymphoid changes did not occur in experimentally immunosuppressed animals, incriminating the immune system in development of lesions (Reardon and Pierce, 1981b). Harrus et al. (1998) demonstrated that the spleen plays an important role in the pathogenesis of CME and proposed that splenic substances and/or inflammatory mediators are involved. Ascites in two animals could have resulted from vasculitis or hypoalbminemia associated with hypergamaglobulinemia since the infected dogs showed total protein levels within normal ranges (Van Dijk, 1971; Simpson, 1974; Hoskins, 1991). Hepatic, renal and CNS lesions may have occurred due to compressive circulatory and/or immunomediated alterations. Hepatic lesions have been reported by Reardon and Pierce (1981a) in dogs with canine ehrlichiosis. Vasculitis was a consistent finding in tissues of all infected animals. It is possible that the general inflammation observed in the vessels (vasculitis) contributes to the pathogenesis of the disease (Van Dijk, 1971; Simpson, 1974). Immunohistochemistry of lymphoid organs revealed increased number of IgM and IgG immunolabeled cells in dogs with CME. Such findings associated with an increase in specific anti-E. canis IgG antibody titers, indicate that humoral responses may play an important role in CME (Cadman et al., 1994). IgG and IgM immunolabeled cell populations are, at least in part, responsible for the morphological changes observed in the spleen and lymph nodes. This observation was also suggested by Reardon and Pierce (1981a), even though these authors did not perform immunohistochemical analysis. In humoral immune response to E. canis IgG2 is predominant in natural and experimental infection (Harrus et al., 2001). Anti-E. canis-IgG2 serum levels were found to be higher than anti-E. canis-IgG1 during the different phases of the disease in symptomatic and asymptomatic dogs (Harrus et al., 2001). IgG immunoglobulins are known to activate complement and thus participate in type III hypersensitivity reactions (Abbas et al., 2000). It is tempting to speculate that such mechanism have a role in the vasculitis observed in CME. CD3+ cells were present in higher numbers in the splenic cords and in the medullary region of lymph nodes, and in lower numbers in the follicular region of the lymph nodes of all infected dogs when compared to control animals. The lower number of CD3+ cells in the follicular region of the lymph nodes may represent an important finding. CD3+ cells found in follicular areas are probably T helper CD4+ cells (Caswell et al., 1995). Based on this observation, a decreased number of T helper cells may be indicative of immunological dysfunction in dogs with CME. Unfortunately, CD4+ T cells could not be immunophenotyped by the time of the conduct of this work by the lack of specific immune reagents in our laboratory. In this regard, an additional issue to be studied in future experimental projects of CME pathogenesis would be the cytokine profile produced by CD4+ cells during infection. It is already well-established that different subsets of CD4+ cells (Th1 or Th2 ) may be responsible for very different outcome in infectious diseases (Mosmann and Sad, 1996; Meusen, 1998). CD8+ cell populations were increased in the follicular and medullary regions of the lymph nodes of animals infected with E. canis. This finding demonstrates that cell-mediated responses were pronounced in inoculated dogs. Kakoma et al. (1977) and Nyindo et al. (1980) reported the importance of cellular immune response in the clinical development
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of the disease. Our finding supports observations made by Page (1995) who reported an increased number of CD8+ cells in peripheral blood of dogs with canine ehrlichiosis. On the other hand, CD8+ cells were decreased in the spleen of infected dogs which could have occurred as an immune response imbalance in this organ in CME. CD8+ cells may represent a protective immune response against persistent E. canis that resists eradication by phagocytes and antibodies while it can also mediate tissue injury by cytolytic elimination of parasitized host cells and thus contribute to CME pathogenesis. Broadly, the present study shows an intense involvement of immune cells in canine ehrlichiosis, especially in lymph nodes and spleen. Immune-mediated vasculitis may play a central role in the pathogenesis of CME and could explain most of the important lesions observed in organs and tissues of infected dogs. Severity of lesions in infected animals in the presence of an intense immune activity suggests immune-mediated mechanisms in the genesis of lesions. Additional studies are being conducted in an attempt to elucidate the mechanisms involved in cell-mediated immune responses in CME.
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