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Presence of anti-platelet IgM and IgG antibodies in dogs naturally infected by Leishmania infantum
Veterinary Immunology and Immunopathology 110 (2006) 331–337 www.elsevier.com/locate/vetimm
Presence of anti-platelet IgM and IgG antibodies in dogs naturally infected by Leishmania infantum G. Terrazzano a, L. Cortese b, D. Piantedosi b, S. Zappacosta a, A. Di Loria b, D. Santoro b, G. Ruggiero a, P. Ciaramella b,* a
Chair of Immunology, Department of Cellular and Molecular Biology and Pathology, University of Naples, Federico II, Via Pansini 5, Napoli, Italy b Department of Veterinary Clinical Science, Section of Internal Medicine, University of Naples, Federico II, Via Delpino 1, 80137 Napoli, Italy
Received 29 July 2005; received in revised form 6 October 2005; accepted 3 November 2005
Abstract Thirty-three dogs, naturally infected by Leishmania infantum, were enrolled in the study and were classified as oligosymptomatic (n. 15) and symptomatic or markedly symptomatic (n. 18). A control group was 10 healthy dogs. A haematological profile was obtained and the dogs serum was employed to assess the presence of platelet binding IgM and IgG antibodies (PBIgM, PBIgG) using flow cytometry. FITC labelled goat anti-dog IgM or IgG were used to detect PBIgM and PBIgG. Samples with a mean fluorescence intensity (MFI) that was 100 channels higher on a log scale for more than 30% of the platelets than seen in negative control platelets from a healthy dog were considered positive for the presence of anti-platelet antibodies (PBIg). Twenty-one (63.3%) dogs revealed the presence of PBIg. Six of them were oligo-symptomatic while 15 showed moderate or severe clinical signs of illness. All the dogs with PBIg showed the presence of PBIgM, with nine animals showing both PBIgM and PBIgG. Nine of 18 symptomatic or markedly symptomatic dogs showed thrombocytopenia, while normal platelet counts were observed in all oligo-symptomatic animals. Eight of 9 thrombocytopenic animals showed the presence of PBIgM, while six of them showed PBIgG. One thrombocytopenic dog was negative for PBIg. This study is the first report documenting the presence of PBIg in natural canine leishmaniasis implying a pathogenic association between thrombocytopenia and the presence of antibody against platelet membrane. # 2005 Elsevier B.V. All rights reserved. Keywords: Canine leishmaniasis; Platelet-bound antibodies; Secondary immune-mediated thrombocytopenia; Flow cytometry
Abbreviations: CL, canine leishmaniasis; CIC, circulating immune complex; PBIg, platelet-bound antibodies; IMT, immune-mediated thrombocytopenia; PRP, platelet-rich plasma; MFI, mean fluorescence intensity; ADP, adenosine 50 -diphosphate * Corresponding author. Tel.: +39 081 2536009; fax: +39 081 2536008. E-mail address: [email protected] (P. Ciaramella).
1. Introduction Canine leishmaniasis (CL) is a zoonotic disease caused by the protozoan Leishmania infantum. The disease is endemic in the Mediterranean area where
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the infected dogs constitute the most important reservoir of parasite infection (WHO, 1990). The clinical features of the CL are widely different as a result of the numerous pathogenic and immunological changes observed in the disease (Ferrer, 1992; Ciaramella and Corona, 2003). Dogs with leishmaniasis often show clinical signs of bleeding such as epistaxis, haematuria and haemorrhagic diarrhoea (Ferrer et al., 1991; Ciaramella et al., 1997; Koutinas et al., 1999). The pathogenesis of bleeding is uncertain and many authors suggested that it is caused by the presence of ulcerations and/or a haemorrhagic diathesis followed to hyperglobulinaemia, paraproteinaemia, and azotaemia and/or primary or secondary haemostasis alteration (Slappendel and Ferrer, 1998). In our previous studies, thrombocytopenia has been reported in 29.3% of CL cases while a deficiency in platelet aggregation have been found in all infected dogs (Ciaramella et al., 1997, 2005). Thrombocytopenia and thrombocytopatia may result from abnormal vascular wall due to vasculitis, altered thrombocytopoiesis, increase in platelet destruction and/or following to renal or hepatic failure (Ferrer, 1992; Slappendel and Ferrer, 1998; Ciaramella et al., 2005). An immunological component has been also suspected in CL following the presence of platelet-bound antibodies (PBIg) in kala-azar patients (Kharazmi et al., 1982; Ciaramella et al., 2005). Indeed, infected dogs show high serum concentration in circulating immune complex (CIC), whose role in platelet disturbances remains speculative. The increased formation and deposition of CIC could contribute to both vascular damage and platelet membrane alteration resulting in a primary impairment of haemostasis. Besides secondary immune-mediated thrombocytopenia (IMT) have been suspected in CL (Chabanne et al., 2000). In secondary IMT, platelet destruction could result from immune complex bound to platelet Fc receptors or new antigens generated during the course of an infection disease (Lewis and Meyers, 1996). The principal antibody class associated with IMT is IgG, although other antibodies classes have been implicated (Lewis et al., 1995). Flow cytometry methods is actually considered one of the most specific and sensitive laboratory test to reveal PBIg in canine thrombocytopenic disorders (Chabanne et al., 2000).
The objective of this study is to evaluate the presence of antibodies against platelet in dogs naturally infected by L. infantum with different grades of clinical involvement, and in dogs showing thrombocytopenia, in order to improve knowledge of the biological mechanisms underlying the platelet disorders frequently associated with this protozoan disease.
2. Materials and methods 2.1. Animals Thirty-three (18 males, 15 females) dogs of mixed breeds, naturally infected by L. infantum, were enrolled in the study. Clinical diagnosis was always confirmed by the detection of amastigotes in lymph nodes or bone marrow aspirate smears and positive IFAT serology. In this context, a threshold titre of 1/ 160 was considered indicative of infection. All of the dogs were serologically negative for Erhlichia canis and had not received any anti-leishmania therapy. According to clinical features, the enrolled animals (Table 1) were classified as oligo-symptomatic (n. 15), symptomatic or markedly symptomatic (n. 18), following the clinical classification reported in a previous study (Ciaramella et al., 1997). Ten clinically healthy dogs, negative for CL and E. canis, were used as controls through the study. All the experiments were approved according to national ethical guidelines. 2.2. Sample collection and haematological evaluations Blood samples, always performed from overnight fasted dogs, were transferred to plastic tubes containing sodium citrate (3.8%). A full blood count was performed within 30 min from the collection, using a semi-automatic cell counter (Genius S; SEAC Radom Group, Florence, Italy). The platelet count assessment was always confirmed by using May-Gru¨enwaldGiemsa stained blood smears. Normal platelet reference ranges considered was 200,000–500,000 cells/ml (Meinkoth and Clinkenbeard, 2000). Serum aliquots were obtained from all the dogs enrolled in the study by centrifugation within 1 h from
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Table 1 Parts A and B show haematological data and PBIgM and PBIgG serum level from oligosymptomatic and symptomatic or markedly symptomatic animals, respectively WBC (103 ml1)
RBC (106 ml1)
Part A: oligosymptomatic group #1 8.9 6.8 #2 11.1 6.9 #3 10.7 6.5 #4 5.9 7.0 #5 11.2 7.0 #6 5.8 7.7 #7 11.6 6.7 #8 11.0 5.7 #9 13.0 6.1 #10 9.2 6.7 #11 8.9 6.6 #12 11.8 5.3 #13 10.6 5.6 #14 11.0 7.2 #15 8.3 6.9
HGB (g/l)
HCT (%)
MCV (fl)
MCH (pg)
MCHC (g/l)
PLT (109 l1)
PBIgM
PBIgG
18.1 17.8 16.7 17.4 17.1 18.5 16.8 11.4 10.6 16.1 17.2 13.2 13.8 12.9 16.7
47.9 44.8 43.7 46.0 47.6 53.1 46.0 39.3 39.6 44.2 43.8 35.9 36.7 46.6 46.1
69 64 67 65 67 69 68 68 65 65 66 67 65 65 67
26.3 25.0 25.6 24.7 24.3 24.0 24.9 19.9 17.0 23.8 26.1 24.8 24.5 28.5 24.4
37.9 39.7 38.1 37.8 36.0 34.9 36.6 29.1 26.8 36.5 39.4 36.8 37.6 43.9 36.2
221 211 339 238 238 271 351 368 393 220 320 246 248 293 256
+a + + + + +
+
22.7 42.6 37.2 40.1 43.5 22.3 23.0 29.2 28.9 36.4 41.0 42.4 35.9 35.2 44.1 39.9 35.9 38.1
68 70 65 70 70 58 56 65 65 67 64 63 67 67 64 68 67 66
25.6 23.6 22.2 24.8 24.7 24.2 19.3 22.9 23.1 18.5 23.2 23.1 24.8 24.5 23.6 23.4 24.8 23.5
37.6 33.7 34.2 35.2 35.3 41.6 34.5 35.1 35.5 27.5 36.0 36.4 36.8 36.6 37.1 34.6 36.8 35.8
88 128 142 79 114 134 115 101 95 210 206 200 210 210 263 286 246 290
+ ++a + + ++ ++ + ++ + + ++ + ++ + +
+ + + ++ ++ + + +
Part B: symptomatic and markedly symptomatic dogs #16 6.4 3.3 8.5 #17 10.5 6.0 14.4 #18 7.2 5.7 12.7 #19 8.8 5.7 14.1 #20 11.3 6.2 15.4 #21 6.2 3.8 9.3 #22 11.5 4.1 7.9 #23 7.9 4.5 10.2 #24 10.0 4.4 10.3 #25 9.1 5.4 10.0 #26 7.0 6.3 14.8 #27 8.7 6.6 15.5 #28 5.8 5.3 13.2 #29 11.5 5.3 12.9 #30 9.3 6.9 16.4 #31 10.5 5.9 13.8 #32 16.8 5.3 13.2 #33 8.2 5.7 10.5
The first nine animals of part B (#16–24) show thrombocytopenia. a Samples with more 30% or 50% of platelets showing a MFI of 100 intensity channels on long scale higher than negative control, are indicated with (+) and (++), respectively.
the sample collection and stored at 70 8C until the assessment of their platelet binding ability by flow cytometry. 2.3. Platelet isolation and immune-fluorescence assay Blood samples, collected into EDTA, were obtained from one healthy dog donor. The plate-
let-rich plasma (PRP) fraction was purified by centrifugation at 300 g for 10 min at 20 8C, as previously described (Kristensen et al., 1994a). After collection to within 0.5 cm of the red blood cell layer, the PRP was transferred into a plastic tube and washed three times with PBS containing 3 mM EDTA and 1% bovine serum albumin (PBS-EDTABSA). After the wash the platelets were adjusted to a concentration of 4 108 cells/ml and used in the
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immune fluorescence assay. Freshly prepared platelets were used each time. Twenty microliters of the platelet suspension were incubated in plastic tubes (Falcon, Becton Dickinson, Mountain View, CA) for 30 min at room temperature with 20 ml of a 1:10 dilution of the sera. After two washes with PSB-EDTA-BSA, platelets were incubated for 30 min with 1:20 FITC labelled goat antidog IgM or IgG (Bethyl Laboratoires, Inc., TX). The platelet population gated by using forward scatter (FSC) and side scatter (SSC) parameters, as described (Kristensen et al., 1994a). Flow cytometry and data analysis, were performed by using a two laser equipped FACScalibur apparatus and the CellQuest analysis software (Becton Dickinson, Mountain View, CA). In order to establish a reference negative value, platelet staining profiles with platelets obtained from 10 healthy animals, was assessed. Only the dog samples with more than 30% of platelets showing a mean fluorescence intensity (MFI) on a log scale that was 100 channels higher than the negative control
value obtained with platelets incubated with healthy dog sera were considered as positive indicating the presence of platelet binding IgM and IgG antibodies (PBIgM or PBIgG). 2.4. Statistical analysis The statistical analysis was performed by using Fisher’s exact test with two-side p value. Results were considered significant when a p < 0.05 was obtained.
3. Results 3.1. Analysis of haematological parameters in leishmania infected dogs Haematological evaluation of infected dogs is reported in Table 1. Briefly, normochromic and normocytic anemia was observed in two and seven oligosymptomatic and symptomatic or markedly
Fig. 1. Isotype analysis of PBIg in the plasma of naturally infected leishmania dogs. Staining profiles of healthy donor platelets treated with FITC-labeled anti-IgM (panels A and B) or anti-IgG (panels C and D) after incubation with healthy dog (panels A and C) or leishmania infected plasma specimens (B and D). The dotted line indicates the staining profiles obtained without plasma treatment. Reported data are representative of the platelet binding activity observed in control animals as compared with one leishmania infected dog. See Section 2 for details.
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symptomatic dogs respectively. Nine of 18 symptomatic or markedly symptomatic dogs showed low platelet count levels, while normal platelet range was observed in all oligosymptomatic animals (Table 1) (P = 0.0014). All the healthy animals included in the study showed a normal haematological profile (not shown). 3.2. Analysis of platelet binding antibodies in the plasma of leishmania affected dogs Fig. 1 shows a negative platelet binding profile, obtained by using the plasma sample of a healthy dog, and a clear positive binding obtained in the presence of a plasma specimen from a leishmania infected dog. Table 1 summarises the results obtained for PBIgM or PBIgG occurrence in the group of naturally infected dogs. No binding was observed after platelet incubation with the plasma specimens from the 10 healthy animals (control group) (Fig. 1). As shown in Table 1, the presence of PBIgM or PBIgM and PBIgG antibodies was observed in a significant percentage of the plasma samples belonging to the leishmania infected dogs. Indeed, 21 (63.3%) of 33 leishmania infected dogs revealed the presence of detectable PBIg. Moreover, 6 of 21 infected animals (18.1%) were oligo-symptomatic while 15 (45.4%) showed moderate or severe clinical signs of illness. These results indicate a significant association between the occurrence of leishmania infection and the presence of anti-platelet immunoglobulins in naturally infected dogs (P = 0.0234). In addition, a significant association between the clinical disease stage and PBIg was also observed (P = 0.0143). The isotype analysis of the platelet bound immunoglobulins revealed that all the dogs with detectable anti-platelet immunoglobulins showed the presence of PBIgM, with nine animals showing both PBIgM and PBIgG (42.8%). In addition, one of the nine animals with contemporary presence of PBIgM and PBIgG was oligo-symptomatic while the remaining eight animals were symptomatic or markedly symptomatic dogs (P = 0.0214). Notably, eight (88.8%) of nine thrombocytopenic animals showed the presence of PBIgM while six of them (66.6%) revealed the occurrence of PBIgG. One thrombocytopenic dog was negative for both IgM and IgG antiplatelet antibodies.
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4. Discussion This study reveals the occurrence of PBIgM as well as IgG isotype in naturally leishmania-infected dogs. The presence of antibodies to platelet in the plasma of infected animals has been also observed to be significantly associated with the clinical stage of the disease and with the presence of a low platelet count. The association between a Th2 type immune response and CL has been already described (Pinelli et al., 1999). Indeed, the occurrence of consistent polyclonal B-cell response leading to non-specific and specific antibody production against the parasite antigens has been extensively demonstrated (Pinelli et al., 1994, 1999). In this context, the presence of an ineffective antibody production has been suggested to be likely responsible for several autoimmune phenomena such the presence of CIC, of rheumatoid factor and of anti-nuclear antibody (Slappendel, 1988). Autoimmune disease has been described in CL (Ginel et al., 1993) but no association between the incidence of auto-antibodies and the clinical stage of the disease have been reported (Ciaramella et al., 1997). On the contrary, this study reveals an association between the presence of platelet binding antibodies and the clinical stage of the disease. Indeed, 15 dogs between the 21 animals showing the occurrence of PBIg were symptomatic or markedly symptomatic while only 6 showed few signs of the disease. The possible involvement of recurrent natural leishmania infections is expected to account for the presence of both IgM and IgG anti-platelet antibodies in the infected dogs. In addition, the occurrence of thymus independent B cell responses eventually driven by leishmania antigens in the naturally infected dogs could explain the preferential presence of IgM anti-platelet antibodies (Wabl and Steinberg, 1996; Schofield, 1991). The presence of anti-platelet auto-antibodies or antibodies bound to platelet antigens altered during the course of illness could account for the occurrence of PBIg by us revealed. In this regard, Dominiquez and Torano (2001) suggested that in the first phases of infection the leishmania parasites are able to directly interact with the platelets by a specific mechanism termed ‘‘immune adherence’’, with the formation of large aggregates. It is also possible that during this
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phase the parasite could modify platelet membranes, in this way favouring their recognition by immunemediated processes. Moreover, the possibility that PBIg could be also represented by IgM or IgG-circulating immune complex bound to Fc platelet receptors has to be considered. In this context, an increased level of IgM or IgG-circulating immune complex has been reported in leishmania infected dogs and the occurrence of associated autoimmune disorders has been also referred (Lopez et al., 1996; Margarito et al., 1998). Previous our findings showed a decrease in platelet aggregation in all leishmania infected dogs, strongly correlated with clinical phase of the disease, especially when collagen was employed as agonist (Pelagalli et al., 2004; Ciaramella et al., 2005). The greater sensitivity of collagen compared with adenosine 50 -diphosphate (ADP) was attributed to the relative differences in membrane receptor affinity, potentially altered by immune-mediated process elicited by parasite. Indeed, ADP and collagen interact essentially with GPIIb/IIIa and GPIa/IIa platelet receptors, respectively (Born, 1962; Coller et al., 1989). In human patient with thrombocytopenia, Kiefel et al. (1996) reported a high affinity between GPIIb/IIIa platelet membrane receptor and PBIgG. In the light of these findings, we speculate that lower PBIgG might cause minimum alterations of GPIIb/IIIa receptor and, consequently, a better activity of ADP. However, future studies are in progress to value the membrane platelet receptor modifications in CL. Our data suggest that PBIg could underlie an immune pathogenesis for thrombocytopenia inCL. In this context, thrombocytopenia associated with dirofilariasis, ehrlichiosis, B. canis and Rickettsii infection in dogs has been also suggested to recognise an immune-mediated pathogenesis (Cockburn and Troy, 1986; Troy and Forrester, 1990; Breitshwerdt, 1988, 1990). In this study, the occurrence of secondary IMT could be also considered. The 66.6% (6/9 dogs) show both PBIgM and PBIgG while 22.2% (2/9 dogs) reveal the presence only of IgM anti-platelet antibodies. In humans, the presence of PBIgG and PBIgM has been observed in 30% of IMT cases (Aster, 1989; Tijhuis et al., 1991), while high affinity PBIgG has been detected in the serum of 55–80% of IMT dog cases (Campbell et al., 1984; Kristensen et al., 1994a,b). In addition, the occurrence of other immunoglobulin
classes has been also suggested (Lewis et al., 1995). Different sensibility of the laboratory tests employed could account for the differences observed in these studies. However, the absence of PBIg in one dog (#16) with thrombocytopenia suggests multiple aetiology. Hypersplenism, renal failure, altered myelopoiesis and systemic vasculitis have also been evoked as important factors in determining the degree of thrombocytopenia in canine leishmaniasis (Ferrer, 1992; Slappendel and Ferrer, 1998; Russell and Grindem, 2000). In conclusion this study, at our best knowledge, is the first report documenting the presence of PBIgM and PBIgG in naturally canine leishmaniasis also showing a pathogenic association between thrombocytopenia and the presence of antibody against platelet membrane in naturally occurring CL.
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Presence of anti-platelet IgM and IgG antibodies in dogs naturally infected by Leishmania infantum G. Terrazzano a, L. Cortese b, D. Piantedosi b, S. Zappacosta a, A. Di Loria b, D. Santoro b, G. Ruggiero a, P. Ciaramella b,* a
Chair of Immunology, Department of Cellular and Molecular Biology and Pathology, University of Naples, Federico II, Via Pansini 5, Napoli, Italy b Department of Veterinary Clinical Science, Section of Internal Medicine, University of Naples, Federico II, Via Delpino 1, 80137 Napoli, Italy
Received 29 July 2005; received in revised form 6 October 2005; accepted 3 November 2005
Abstract Thirty-three dogs, naturally infected by Leishmania infantum, were enrolled in the study and were classified as oligosymptomatic (n. 15) and symptomatic or markedly symptomatic (n. 18). A control group was 10 healthy dogs. A haematological profile was obtained and the dogs serum was employed to assess the presence of platelet binding IgM and IgG antibodies (PBIgM, PBIgG) using flow cytometry. FITC labelled goat anti-dog IgM or IgG were used to detect PBIgM and PBIgG. Samples with a mean fluorescence intensity (MFI) that was 100 channels higher on a log scale for more than 30% of the platelets than seen in negative control platelets from a healthy dog were considered positive for the presence of anti-platelet antibodies (PBIg). Twenty-one (63.3%) dogs revealed the presence of PBIg. Six of them were oligo-symptomatic while 15 showed moderate or severe clinical signs of illness. All the dogs with PBIg showed the presence of PBIgM, with nine animals showing both PBIgM and PBIgG. Nine of 18 symptomatic or markedly symptomatic dogs showed thrombocytopenia, while normal platelet counts were observed in all oligo-symptomatic animals. Eight of 9 thrombocytopenic animals showed the presence of PBIgM, while six of them showed PBIgG. One thrombocytopenic dog was negative for PBIg. This study is the first report documenting the presence of PBIg in natural canine leishmaniasis implying a pathogenic association between thrombocytopenia and the presence of antibody against platelet membrane. # 2005 Elsevier B.V. All rights reserved. Keywords: Canine leishmaniasis; Platelet-bound antibodies; Secondary immune-mediated thrombocytopenia; Flow cytometry
Abbreviations: CL, canine leishmaniasis; CIC, circulating immune complex; PBIg, platelet-bound antibodies; IMT, immune-mediated thrombocytopenia; PRP, platelet-rich plasma; MFI, mean fluorescence intensity; ADP, adenosine 50 -diphosphate * Corresponding author. Tel.: +39 081 2536009; fax: +39 081 2536008. E-mail address: [email protected] (P. Ciaramella).
1. Introduction Canine leishmaniasis (CL) is a zoonotic disease caused by the protozoan Leishmania infantum. The disease is endemic in the Mediterranean area where
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the infected dogs constitute the most important reservoir of parasite infection (WHO, 1990). The clinical features of the CL are widely different as a result of the numerous pathogenic and immunological changes observed in the disease (Ferrer, 1992; Ciaramella and Corona, 2003). Dogs with leishmaniasis often show clinical signs of bleeding such as epistaxis, haematuria and haemorrhagic diarrhoea (Ferrer et al., 1991; Ciaramella et al., 1997; Koutinas et al., 1999). The pathogenesis of bleeding is uncertain and many authors suggested that it is caused by the presence of ulcerations and/or a haemorrhagic diathesis followed to hyperglobulinaemia, paraproteinaemia, and azotaemia and/or primary or secondary haemostasis alteration (Slappendel and Ferrer, 1998). In our previous studies, thrombocytopenia has been reported in 29.3% of CL cases while a deficiency in platelet aggregation have been found in all infected dogs (Ciaramella et al., 1997, 2005). Thrombocytopenia and thrombocytopatia may result from abnormal vascular wall due to vasculitis, altered thrombocytopoiesis, increase in platelet destruction and/or following to renal or hepatic failure (Ferrer, 1992; Slappendel and Ferrer, 1998; Ciaramella et al., 2005). An immunological component has been also suspected in CL following the presence of platelet-bound antibodies (PBIg) in kala-azar patients (Kharazmi et al., 1982; Ciaramella et al., 2005). Indeed, infected dogs show high serum concentration in circulating immune complex (CIC), whose role in platelet disturbances remains speculative. The increased formation and deposition of CIC could contribute to both vascular damage and platelet membrane alteration resulting in a primary impairment of haemostasis. Besides secondary immune-mediated thrombocytopenia (IMT) have been suspected in CL (Chabanne et al., 2000). In secondary IMT, platelet destruction could result from immune complex bound to platelet Fc receptors or new antigens generated during the course of an infection disease (Lewis and Meyers, 1996). The principal antibody class associated with IMT is IgG, although other antibodies classes have been implicated (Lewis et al., 1995). Flow cytometry methods is actually considered one of the most specific and sensitive laboratory test to reveal PBIg in canine thrombocytopenic disorders (Chabanne et al., 2000).
The objective of this study is to evaluate the presence of antibodies against platelet in dogs naturally infected by L. infantum with different grades of clinical involvement, and in dogs showing thrombocytopenia, in order to improve knowledge of the biological mechanisms underlying the platelet disorders frequently associated with this protozoan disease.
2. Materials and methods 2.1. Animals Thirty-three (18 males, 15 females) dogs of mixed breeds, naturally infected by L. infantum, were enrolled in the study. Clinical diagnosis was always confirmed by the detection of amastigotes in lymph nodes or bone marrow aspirate smears and positive IFAT serology. In this context, a threshold titre of 1/ 160 was considered indicative of infection. All of the dogs were serologically negative for Erhlichia canis and had not received any anti-leishmania therapy. According to clinical features, the enrolled animals (Table 1) were classified as oligo-symptomatic (n. 15), symptomatic or markedly symptomatic (n. 18), following the clinical classification reported in a previous study (Ciaramella et al., 1997). Ten clinically healthy dogs, negative for CL and E. canis, were used as controls through the study. All the experiments were approved according to national ethical guidelines. 2.2. Sample collection and haematological evaluations Blood samples, always performed from overnight fasted dogs, were transferred to plastic tubes containing sodium citrate (3.8%). A full blood count was performed within 30 min from the collection, using a semi-automatic cell counter (Genius S; SEAC Radom Group, Florence, Italy). The platelet count assessment was always confirmed by using May-Gru¨enwaldGiemsa stained blood smears. Normal platelet reference ranges considered was 200,000–500,000 cells/ml (Meinkoth and Clinkenbeard, 2000). Serum aliquots were obtained from all the dogs enrolled in the study by centrifugation within 1 h from
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Table 1 Parts A and B show haematological data and PBIgM and PBIgG serum level from oligosymptomatic and symptomatic or markedly symptomatic animals, respectively WBC (103 ml1)
RBC (106 ml1)
Part A: oligosymptomatic group #1 8.9 6.8 #2 11.1 6.9 #3 10.7 6.5 #4 5.9 7.0 #5 11.2 7.0 #6 5.8 7.7 #7 11.6 6.7 #8 11.0 5.7 #9 13.0 6.1 #10 9.2 6.7 #11 8.9 6.6 #12 11.8 5.3 #13 10.6 5.6 #14 11.0 7.2 #15 8.3 6.9
HGB (g/l)
HCT (%)
MCV (fl)
MCH (pg)
MCHC (g/l)
PLT (109 l1)
PBIgM
PBIgG
18.1 17.8 16.7 17.4 17.1 18.5 16.8 11.4 10.6 16.1 17.2 13.2 13.8 12.9 16.7
47.9 44.8 43.7 46.0 47.6 53.1 46.0 39.3 39.6 44.2 43.8 35.9 36.7 46.6 46.1
69 64 67 65 67 69 68 68 65 65 66 67 65 65 67
26.3 25.0 25.6 24.7 24.3 24.0 24.9 19.9 17.0 23.8 26.1 24.8 24.5 28.5 24.4
37.9 39.7 38.1 37.8 36.0 34.9 36.6 29.1 26.8 36.5 39.4 36.8 37.6 43.9 36.2
221 211 339 238 238 271 351 368 393 220 320 246 248 293 256
+a + + + + +
+
22.7 42.6 37.2 40.1 43.5 22.3 23.0 29.2 28.9 36.4 41.0 42.4 35.9 35.2 44.1 39.9 35.9 38.1
68 70 65 70 70 58 56 65 65 67 64 63 67 67 64 68 67 66
25.6 23.6 22.2 24.8 24.7 24.2 19.3 22.9 23.1 18.5 23.2 23.1 24.8 24.5 23.6 23.4 24.8 23.5
37.6 33.7 34.2 35.2 35.3 41.6 34.5 35.1 35.5 27.5 36.0 36.4 36.8 36.6 37.1 34.6 36.8 35.8
88 128 142 79 114 134 115 101 95 210 206 200 210 210 263 286 246 290
+ ++a + + ++ ++ + ++ + + ++ + ++ + +
+ + + ++ ++ + + +
Part B: symptomatic and markedly symptomatic dogs #16 6.4 3.3 8.5 #17 10.5 6.0 14.4 #18 7.2 5.7 12.7 #19 8.8 5.7 14.1 #20 11.3 6.2 15.4 #21 6.2 3.8 9.3 #22 11.5 4.1 7.9 #23 7.9 4.5 10.2 #24 10.0 4.4 10.3 #25 9.1 5.4 10.0 #26 7.0 6.3 14.8 #27 8.7 6.6 15.5 #28 5.8 5.3 13.2 #29 11.5 5.3 12.9 #30 9.3 6.9 16.4 #31 10.5 5.9 13.8 #32 16.8 5.3 13.2 #33 8.2 5.7 10.5
The first nine animals of part B (#16–24) show thrombocytopenia. a Samples with more 30% or 50% of platelets showing a MFI of 100 intensity channels on long scale higher than negative control, are indicated with (+) and (++), respectively.
the sample collection and stored at 70 8C until the assessment of their platelet binding ability by flow cytometry. 2.3. Platelet isolation and immune-fluorescence assay Blood samples, collected into EDTA, were obtained from one healthy dog donor. The plate-
let-rich plasma (PRP) fraction was purified by centrifugation at 300 g for 10 min at 20 8C, as previously described (Kristensen et al., 1994a). After collection to within 0.5 cm of the red blood cell layer, the PRP was transferred into a plastic tube and washed three times with PBS containing 3 mM EDTA and 1% bovine serum albumin (PBS-EDTABSA). After the wash the platelets were adjusted to a concentration of 4 108 cells/ml and used in the
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immune fluorescence assay. Freshly prepared platelets were used each time. Twenty microliters of the platelet suspension were incubated in plastic tubes (Falcon, Becton Dickinson, Mountain View, CA) for 30 min at room temperature with 20 ml of a 1:10 dilution of the sera. After two washes with PSB-EDTA-BSA, platelets were incubated for 30 min with 1:20 FITC labelled goat antidog IgM or IgG (Bethyl Laboratoires, Inc., TX). The platelet population gated by using forward scatter (FSC) and side scatter (SSC) parameters, as described (Kristensen et al., 1994a). Flow cytometry and data analysis, were performed by using a two laser equipped FACScalibur apparatus and the CellQuest analysis software (Becton Dickinson, Mountain View, CA). In order to establish a reference negative value, platelet staining profiles with platelets obtained from 10 healthy animals, was assessed. Only the dog samples with more than 30% of platelets showing a mean fluorescence intensity (MFI) on a log scale that was 100 channels higher than the negative control
value obtained with platelets incubated with healthy dog sera were considered as positive indicating the presence of platelet binding IgM and IgG antibodies (PBIgM or PBIgG). 2.4. Statistical analysis The statistical analysis was performed by using Fisher’s exact test with two-side p value. Results were considered significant when a p < 0.05 was obtained.
3. Results 3.1. Analysis of haematological parameters in leishmania infected dogs Haematological evaluation of infected dogs is reported in Table 1. Briefly, normochromic and normocytic anemia was observed in two and seven oligosymptomatic and symptomatic or markedly
Fig. 1. Isotype analysis of PBIg in the plasma of naturally infected leishmania dogs. Staining profiles of healthy donor platelets treated with FITC-labeled anti-IgM (panels A and B) or anti-IgG (panels C and D) after incubation with healthy dog (panels A and C) or leishmania infected plasma specimens (B and D). The dotted line indicates the staining profiles obtained without plasma treatment. Reported data are representative of the platelet binding activity observed in control animals as compared with one leishmania infected dog. See Section 2 for details.
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symptomatic dogs respectively. Nine of 18 symptomatic or markedly symptomatic dogs showed low platelet count levels, while normal platelet range was observed in all oligosymptomatic animals (Table 1) (P = 0.0014). All the healthy animals included in the study showed a normal haematological profile (not shown). 3.2. Analysis of platelet binding antibodies in the plasma of leishmania affected dogs Fig. 1 shows a negative platelet binding profile, obtained by using the plasma sample of a healthy dog, and a clear positive binding obtained in the presence of a plasma specimen from a leishmania infected dog. Table 1 summarises the results obtained for PBIgM or PBIgG occurrence in the group of naturally infected dogs. No binding was observed after platelet incubation with the plasma specimens from the 10 healthy animals (control group) (Fig. 1). As shown in Table 1, the presence of PBIgM or PBIgM and PBIgG antibodies was observed in a significant percentage of the plasma samples belonging to the leishmania infected dogs. Indeed, 21 (63.3%) of 33 leishmania infected dogs revealed the presence of detectable PBIg. Moreover, 6 of 21 infected animals (18.1%) were oligo-symptomatic while 15 (45.4%) showed moderate or severe clinical signs of illness. These results indicate a significant association between the occurrence of leishmania infection and the presence of anti-platelet immunoglobulins in naturally infected dogs (P = 0.0234). In addition, a significant association between the clinical disease stage and PBIg was also observed (P = 0.0143). The isotype analysis of the platelet bound immunoglobulins revealed that all the dogs with detectable anti-platelet immunoglobulins showed the presence of PBIgM, with nine animals showing both PBIgM and PBIgG (42.8%). In addition, one of the nine animals with contemporary presence of PBIgM and PBIgG was oligo-symptomatic while the remaining eight animals were symptomatic or markedly symptomatic dogs (P = 0.0214). Notably, eight (88.8%) of nine thrombocytopenic animals showed the presence of PBIgM while six of them (66.6%) revealed the occurrence of PBIgG. One thrombocytopenic dog was negative for both IgM and IgG antiplatelet antibodies.
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4. Discussion This study reveals the occurrence of PBIgM as well as IgG isotype in naturally leishmania-infected dogs. The presence of antibodies to platelet in the plasma of infected animals has been also observed to be significantly associated with the clinical stage of the disease and with the presence of a low platelet count. The association between a Th2 type immune response and CL has been already described (Pinelli et al., 1999). Indeed, the occurrence of consistent polyclonal B-cell response leading to non-specific and specific antibody production against the parasite antigens has been extensively demonstrated (Pinelli et al., 1994, 1999). In this context, the presence of an ineffective antibody production has been suggested to be likely responsible for several autoimmune phenomena such the presence of CIC, of rheumatoid factor and of anti-nuclear antibody (Slappendel, 1988). Autoimmune disease has been described in CL (Ginel et al., 1993) but no association between the incidence of auto-antibodies and the clinical stage of the disease have been reported (Ciaramella et al., 1997). On the contrary, this study reveals an association between the presence of platelet binding antibodies and the clinical stage of the disease. Indeed, 15 dogs between the 21 animals showing the occurrence of PBIg were symptomatic or markedly symptomatic while only 6 showed few signs of the disease. The possible involvement of recurrent natural leishmania infections is expected to account for the presence of both IgM and IgG anti-platelet antibodies in the infected dogs. In addition, the occurrence of thymus independent B cell responses eventually driven by leishmania antigens in the naturally infected dogs could explain the preferential presence of IgM anti-platelet antibodies (Wabl and Steinberg, 1996; Schofield, 1991). The presence of anti-platelet auto-antibodies or antibodies bound to platelet antigens altered during the course of illness could account for the occurrence of PBIg by us revealed. In this regard, Dominiquez and Torano (2001) suggested that in the first phases of infection the leishmania parasites are able to directly interact with the platelets by a specific mechanism termed ‘‘immune adherence’’, with the formation of large aggregates. It is also possible that during this
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phase the parasite could modify platelet membranes, in this way favouring their recognition by immunemediated processes. Moreover, the possibility that PBIg could be also represented by IgM or IgG-circulating immune complex bound to Fc platelet receptors has to be considered. In this context, an increased level of IgM or IgG-circulating immune complex has been reported in leishmania infected dogs and the occurrence of associated autoimmune disorders has been also referred (Lopez et al., 1996; Margarito et al., 1998). Previous our findings showed a decrease in platelet aggregation in all leishmania infected dogs, strongly correlated with clinical phase of the disease, especially when collagen was employed as agonist (Pelagalli et al., 2004; Ciaramella et al., 2005). The greater sensitivity of collagen compared with adenosine 50 -diphosphate (ADP) was attributed to the relative differences in membrane receptor affinity, potentially altered by immune-mediated process elicited by parasite. Indeed, ADP and collagen interact essentially with GPIIb/IIIa and GPIa/IIa platelet receptors, respectively (Born, 1962; Coller et al., 1989). In human patient with thrombocytopenia, Kiefel et al. (1996) reported a high affinity between GPIIb/IIIa platelet membrane receptor and PBIgG. In the light of these findings, we speculate that lower PBIgG might cause minimum alterations of GPIIb/IIIa receptor and, consequently, a better activity of ADP. However, future studies are in progress to value the membrane platelet receptor modifications in CL. Our data suggest that PBIg could underlie an immune pathogenesis for thrombocytopenia inCL. In this context, thrombocytopenia associated with dirofilariasis, ehrlichiosis, B. canis and Rickettsii infection in dogs has been also suggested to recognise an immune-mediated pathogenesis (Cockburn and Troy, 1986; Troy and Forrester, 1990; Breitshwerdt, 1988, 1990). In this study, the occurrence of secondary IMT could be also considered. The 66.6% (6/9 dogs) show both PBIgM and PBIgG while 22.2% (2/9 dogs) reveal the presence only of IgM anti-platelet antibodies. In humans, the presence of PBIgG and PBIgM has been observed in 30% of IMT cases (Aster, 1989; Tijhuis et al., 1991), while high affinity PBIgG has been detected in the serum of 55–80% of IMT dog cases (Campbell et al., 1984; Kristensen et al., 1994a,b). In addition, the occurrence of other immunoglobulin
classes has been also suggested (Lewis et al., 1995). Different sensibility of the laboratory tests employed could account for the differences observed in these studies. However, the absence of PBIg in one dog (#16) with thrombocytopenia suggests multiple aetiology. Hypersplenism, renal failure, altered myelopoiesis and systemic vasculitis have also been evoked as important factors in determining the degree of thrombocytopenia in canine leishmaniasis (Ferrer, 1992; Slappendel and Ferrer, 1998; Russell and Grindem, 2000). In conclusion this study, at our best knowledge, is the first report documenting the presence of PBIgM and PBIgG in naturally canine leishmaniasis also showing a pathogenic association between thrombocytopenia and the presence of antibody against platelet membrane in naturally occurring CL.
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