A L V F G a 1 b P a A R R A K L A D S P 1 p c 0 d Veterinary Parasitology 184 (2012) 147– 153 Contents lists available at SciVerse ScienceDirect Veterinary Parasitology jo u rn al hom epa ge : www.elsev ier .com/ locate /vetpar poptosis in T lymphocytes from spleen tissue and peripheral blood of . (L.) chagasi naturally infected dogs aléria Març al Felix de Limaa,∗, Karina Reinaldo Fattorib, Fausto de Souzaa, lavia Rezende Eugênioa, Paulo Sérgio Patto dos Santosa, Daniele Bernadete Rozzaa, isele Fabrino Machadoa Departamento de Clinica, Cirurgia e Reproduç ão Animal, Faculdade de Medicina Veterinária, Universidade Estadual Paulista, Rua Clóvis Pestana, 793, ZIP 6050-400 Araç atuba, São Paulo, Brazil1 Faculdade de Ciências Agrárias e Veterinárias Programa de Pós-graduaç ão em Microbiologia Agropecuária, Universidade Estadual Paulista, Via de Acesso rofessor Paulo Donato Castellane s/n, ZIP 14884-900 Jaboticabal, São Paulo, Brazil r t i c l e i n f o rticle history: eceived 26 November 2010 eceived in revised form 10 August 2011 ccepted 12 August 2011 eywords: eishmaniasis poptosis ogs pleen eripheral blood a b s t r a c t Dogs are the main domestic reservoirs of L. (L.) chagasi. Once in the vertebrate host, the par- asite may cause visceral leishmaniasis, which can also be transmitted to humans. Infected symptomatic dogs show disorganization in the white pulp in spleen tissue and a reduction in T lymphocytes in peripheral blood. To investigate whether apoptosis is involved in white pulp disorganization and diminished T cell counts in peripheral blood, apoptotic T cells from the spleen and peripheral blood of dogs naturally infected with L. (L.) chagasi and presenting clinical manifestations were quantified and compared with healthy dogs. Thirteen symp- tomatic adult dogs infected by L. (L.) chagasi and six healthy dogs from a nonendemic area (controls) were included in the study. Samples from spleen and peripheral blood were used to quantify apoptosis in CD3 lymphocytes by flow cytometry using Anexin V and Multicas- pase kits; the results were compared using the Mann Whitney test. The percentage of total T cells was lower in Leishmania infected dogs compared to healthy controls (P < 0.05). Apo- ptosis levels in T cells from PBMC and spleen were higher in infected dogs than in controls (P < 0.05). The least squares method test was used to determine the effect between the degree of structural organization of spleen white pulp and the percentage of apoptosis in the spleen. A significant effect on the level of white pulp morphological disorganization and percentage of apoptosis in spleen T cells was observed (F = 20.45; P = 0.0014). These data suggest that apoptosis is an important for the immunopathogenesis of canine visceral leishmaniasis. © 2011 Elsevier B.V. All rights reserved. . Introduction Leishmaniasis is caused by a protozoan of the try- anosomatidae family, genus Leishmania. It occurs in 88 ountries of which 65 present the visceral form. Most cases ∗ Corresponding author. Tel.: +55 18 36363285. E-mail address: vmflima@fmva.unesp.br (V.M.F.d. Lima). 1 Tel.: +55 18 3636 14 22. 304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved. oi:10.1016/j.vetpar.2011.08.024 (90%) of visceral leishmaniasis (VL) in humans occur in the rural or suburban areas of five countries, including Brazil (Desjeux, 2004). In Brazil, like other countries in South America, migration into urban areas contributed to the expansion of VL (Desjeux, 2004). In Brazil, this expan- sion occurred especially in the northeast (Dantas-Torres, 2006) and southeast areas (Santiago et al., 2007). Besides the high incidence and broader distribution that the expan- sion itself represents, the spread into new areas also carries the threat that severe and lethal forms of the disease may dx.doi.org/10.1016/j.vetpar.2011.08.024 http://www.sciencedirect.com/science/journal/03044017 http://www.elsevier.com/locate/vetpar mailto:vmflima@fmva.unesp.br dx.doi.org/10.1016/j.vetpar.2011.08.024 ry Paras 148 V.M.F.d. Lima et al. / Veterina emerge when associated with malnutrition (Gontijo and Melo, 2004) and HIV/AIDS infection (Ashford, 2000). Dogs are considered the main domestic reservoirs of L. (L.) chagasi (Moreno and Alvar, 2002). The parasite is transmitted from one infected dog to another through the bite of phlebotominae and possibly through other arthro- pod vectors such as fleas and ticks (Ferreira et al., 2009; Coutinho et al., 2005), and through blood transfusions, as reported by Owens et al. (2001). After the phlebotominae feed on the dogs’ blood, the parasites rapidly spread into the lymph nodes and spleen through the lymph and blood and eventually reach the kidneys and liver. They may also affect the reproductive organs, skin, digestive and respi- ratory systems and the bladder (Molyneux and Ashford, 1983). Once in the vertebrate host, the parasite can cause lesions and symptoms that are characteristic of canine visceral leishmaniasis, although some infected dogs may be oligosymptomatic or asymptomatic (Mancianti and Meciani, 1988), and some may evolve to spontaneous cure (Fisa et al., 1999). The most frequent signs of VL are lymphadenopathy, onychogryphosis, cutaneous lesions, weight lost, cachexia and locomotor abnormali- ties (Semião-Santos et al., 1995). The asymptomatic form represents 20–40% of the serum-positive population, of which 80% actually develops the disease (Noli, 1999). In the Brazil in urban area of the northeast region, the asymptomatic form represent 30% of the serum positive population (Queiroz et al., 2009). The suppression of cellular immunity is the most impor- tant aspect in the pathogenesis and progression of canine disease. The absence of T cell response to antigens of Leish- mania sp. is observed in vivo, with a negative leishmanin skin test (Dos Santos et al., 2008). In dogs infected with Leishmania infantum, a reduction in the number of T lym- phocytes in PBMC occurs (Bourdoiseau et al., 1997) and disorganization of white pulp in spleen tissue has been previously described (Santana et al., 2008), but the mech- anisms that are responsible for these changes have not yet been elucidated. In human acute infection, the reduction in T lympho- cytes and mononuclear cells of peripheral blood and failure in immunity has been associated with apoptosis (Potestio et al., 2004). In mice experimentally infected with Leish- mania donovani, an increase in the level of spontaneous apoptosis in the spleen and liver compared to noninfected mice was also observed (Alexander et al., 2001). In vitro findings also suggest the involvement of apoptosis in the mechanism of suppression observed in visceral leishmani- asis, the infection of macrophages by L. donovani increased the level of FAS in the membrane and sFASL in the cul- ture supernatant, a mechanism that may contribute to increased sensitivity to apoptosis for T cells specific for Leishmania sp. (Eidsmo et al., 2002). To investigate whether apoptosis is involved in the reduction in lymphocytes in peripheral blood and alter- ations in white pulp, apoptosis was quantified in dogs naturally infected with L. (L.) chagasi presenting clinical manifestations and the structural disorganization in white pulp was correlated with the percentage of apoptosis in T cells. If proven, such findings could contribute to improving itology 184 (2012) 147– 153 our present understanding of the immunopathogenesis in infected dogs. 2. Materials and methods 2.1. The study area The county of Araç atuba (21◦12′32′′ S; 50◦25′38′′ W), with an area of 1,167,311 km2, is located in the state of São Paulo, Brazil. It is a region known to be endemic for canine VL. 2.2. Ethical issue This study was approved by the institutional Ethics and Animal Welfare Committee (Comissão de Ética em Experimentaç ão Animal, CEEA, UNESP, process number 02232). 2.3. Animals and diagnosis of VL A total of 13 adult dogs were used, males and females, aged between 2 and 4 years-old, of undefined breed and different weights, from the Zoonosis Control Cen- ter of Araç atuba (CCZA). The dogs were symptomatic and showing at least three clinical signs of canine VL. These could include fever, dermatitis, lymphoadenopathy, onychogryphosis, weight loss, cachexia, locomotor abnor- malities, conjunctivitis, epistaxis, hepatosplenomegaly, edema, and apathy. Briefly, the diagnosis of VL was con- firmed by detecting anti-Leishmania antibodies for L. (L.) chagasi by indirect ELISA, according to Lima et al. (2003), and simultaneously positivity in rapid test rK39 and in PCR amplification of Leishmania spp. DNA in spleen tissue. A group of 6 healthy dogs, both males and females, from a nonendemic area (Londrina, State of Paraná, Brazil) were included in the study as negative controls. These dogs were serum negative for L. (L.) chagasi by indirect ELISA (Lima et al., 2003), negative in the rapid test rK39 and in PCR amplification of Leishmania spp. DNA in spleen tissue. 2.4. Sample collection Samples of spleen from both groups were removed by surgical excision. The dogs were premedicated with the combination of morphine (0.4 mg kg−1 IM) and acepro- mazine (0.05 mg kg−1 IM). Fifteen minutes later, propofol (4.0 mg kg−1 IV) and midazolam (0.1 mg kg−1 IV) were used for induction. Immediately, the dogs were positioned in dorsal recumbency and anesthesia was maintained with isoflurane (1.5 V%). The heart and respiratory rates, the systolic arterial blood pressure and end-tidal CO2 measure- ments were monitored during all anesthetic procedure. The samples were maintained in RPMI 1640 supplemented with 10% (v/v) fetal calf serum (Sigma) at 4 ◦C and processed immediately to evaluate apoptosis. From each dog, 4 ml of blood was collected from the cephalic veins, clotted at room temperature for 4 h and subsequently centrifuged to extract the serum. The serum samples were stored at–20 ◦C prior to analysis and ry Paras a a 2 U b s a 2 f i p s t b t a w E r G C a o P o 1 ( A c 5 1 a − n c p u f 2 a ( l p a t U t t t T c w V.M.F.d. Lima et al. / Veterina dditional blood samples were collected with sodium EDTA nd processed immediately to evaluate apoptosis. .5. Kalazar detect rapid test rK-39 (InBios International, SA) To perform this test, blood samples were collected y venipuncture and centrifuged and the serum was eparated. The procedure of the test was performed in ccordance with the manufacturer’s recommendations. .6. PCR DNA obtained from spleen samples was extracted by reezing and thawing the cells 3 times and washing them n 1× SSC buffer solution (NaCl 3 M, sodium citrate 0.3 M, H 7.0). For cell lysis and protein digestion, 300 �l of lysing olution was added (10% SDS in 0.2 M sodium acetate) ogether with 20 �g/ml proteinase K. Samples were incu- ated at 56 ◦C for 2 h and the DNA was extracted using he phenol/chloroform/isoamyl alcohol method (25:24:1), ccording to Sambrook et al. (1989). After extraction, DNA as resuspended in 50 �l TE (10 mM Tris–HCl pH 8.0, 1 mM DTA pH 8.0) and incubated for 3 min at 60 ◦C. The mate- ial was stored at −20 ◦C until used. The 13A (3′-GTG GGG AG GGG CGT TCT-5′) and 13B (3′-ATT TTA CAC CAA CCC CA GTT-5′) primers were used (Rodgers et al., 1990) to mplify a 120 bp fragment located in the constant region f the kinetoplast minicircles in all Leishmania species. The CR was performed in a 60 �l volume containing 30 pmol f each primer (Invitrogen®), 0.2 mM DNTPs (Invitrogen®), .5 mM MgCl2 (Invitrogen®), 5 U Taq DNA Polymerase Invitrogen®), 50 nM buffer solution, milliQ water and DNA. mplification was performed in an Eppendorf® Mastercy- ler Thermocycler gradient with initial heating to 95 ◦C for min, followed by 33 cycles at 95–57–72 ◦C for 1.5 min, .5 min, and 2 min, respectively. Extension was performed t 72 ◦C for 10 min and the final product was stored at 20 ◦C until analysis. Reaction mixes containing either o DNA or DNA extracted from a L. chagasi promastigote ulture (MHOM/BR00/MER02) were used as negative and ositive controls, respectively. The amplified 120 bp prod- ct was analyzed by electrophoresis on acrylamide gels ollowed by silver staining. .7. Apoptosis evaluation in T cells Peripheral blood cells were stained following the sep- ration of mononuclear cells on a Ficoll-Paque gradient Amersham Biosciences). Spleen cells were processed after ysis of red blood cells. For staining, the cells were sus- ended in PBS containing 1% bovine serum albumin, 0.1% zide and 20% fetal bovine serum to block the Fc recep- or (FCR). Anti-canine CD3 monoclonal antibodies (Serotec, K) were added and incubated for 30 min. Isotype con- rol (Serotec, UK) antibody was added in a separate tube o control for nonspecific labeling. Following incubation, he tubes were centrifuged at 1000 × g for 3 min at 4 ◦C. he supernatant was discarded by quick inversion and the ell pellet briefly vortexed to resuspend the cells. The cells ere washed four times with ice-cold PBS with 10% bovine itology 184 (2012) 147– 153 149 calf sera. After the final wash, the cells were resuspended with PBS. After immune staining for CD3 in PBMC and in leuco- cytes from spleen the apoptosis was detected using two different methods. The Nexin assay, which uses Annexin V, is a calcium-dependent phospholipid binding protein with high affinity for phosphatidylserine, a membrane compo- nent normally located in the internal face; however, during early activation of apoptotic pathways, these molecules are translocated to the outer surface of the cell membrane, where Annexin V can bind directly to them (Vermes et al., 1995). The second method, MultiCaspase SR kit, detects caspase pathways by a fluorescent labeled inhibitor of cas- pase reagent that specifically identifies active caspases. These methods have been used in similar studies (Colgate et al., 2007). The percentage of apoptosis was determined using the Nexin assay Kit (Guava, Hayward, CA) and Mutilcaspase SR kit (Guava, Hayward, CA). The procedure of each test was performed in accordance with the manufacturer’s recom- mendations. Proper instrument performance was verified by running the Guava Check application with Guava Check reagents. Data were acquired regarding the Guava Easy- CyteMini system using CytoSoft software, as described in the Guava PCA User’s Guide and respective package inserts. For Guava MultiCaspase and Guava Nexin, 10,000 events were usually acquired. Negative and positive control Camptothecin (Sigma, USA) (0.15 �g/mL) in DMSO (Sigma) (Tao et al., 2004) were used to verify reagent performance and set analysis markers, delineating the negative and pos- itive populations. 2.8. Spleen samples and histological analysis Three to 4 mm-thick tissue slices were cut transversally to the capsule and fixed in 10% formalin. After fixation, tis- sue slides were embedded in paraffin. Four to 5 �m-thick sections were cut and stained with hematoxylin and eosin (HE). These sections were examined by at least two of the authors who were blind to previous knowledge of the dogs’ health and identities. The authors scored the level of white pulp organization: (1) slightly disorganized, with either hyperplastic or hypoplastic changes leading to a loss of def- inition of any of the regions of the white pulp and (2) for moderately or extensively disorganized, when the white pulp regions were poorly individualized or indistinct. 2.9. Immunohistochemistry (IHC) IHC stains were performed using the standard streptavidin–biotin peroxidase (HRP) immunostaining procedure with polyclonal antibody. Anti-CD3 (A0452) (DAKO, CA, USA) was used to detect T lymphocytes. This antibody was previously used to stain CD3 in canine lym- phomas (Sueiro et al., 2004). Slides were deparaffinized and hydrated. Antigen retrieval was achieved by steam heating in citrate buffer for 30 min. For inhibition of endogenous peroxidase, slides were incubated with 2% (v/v) hydrogen peroxide 30 vol diluted in 50% (v/v) methanol for 30 min and nonspecific binding was blocked with 3% (w/v) nonfat dry milk in PBS for 30 min. Primary antibody against CD3 150 V.M.F.d. Lima et al. / Veterinary Paras Fig. 1. Percentage of CD3 lymphocytes in PBMC from Leishmania infected dogs (INF) and healthy controls (CT). The results are expressed as the mean percentage ± SD. P < 0.05: significant differences between the mean values for Leishmania infected dogs and healthy controls. (1:100) was applied for 18–22 h at 4 ◦C in a humidified chamber. Slides were washed in PBS, incubated with a biotinylated secondary antibody (LSAB+ Kit, DAKO K0690, CA, USA) for 45 min at room temperature, washed once more with PBS, and incubated with streptavidin–HRP complex (LSAB+ Kit, DAKO K0690, CA, USA) for 45 min at room temperature. The reaction was developed with 3,3′- diaminobenzidine (DAKO K3468, CA, USA). The slides were counterstained with Harris’s hematoxylin, dehydrated, cleared and mounted with coverslips. Spleen tissue from healthy dogs was used as a positive control. 2.10. Statistical analysis The data were analyzed by a nonparametric test. Group means were compared using Mann Whitney test. The least squares method was used to evaluate the effect of group (degree of correlation for the structural organiza- tion of white pulp) and quantitative variable (percentage of apoptosis). The results were considered significant when P < 0.05. The SAS software was used (SAS 9.1, SAS Institute, Cary, NC, USA) for all statistical analyses performed in this study. 3. Results 3.1. Decrease in T lymphocytes in PBMC Flow cytometry analysis of CD3 lymphocytes in PMBC from infected dogs showed significantly lower num- bers (58 ± 12, mean ± SD) compared to healthy controls (80.6 ± 5, mean ± SD) (Fig. 1) (P = 0.001, Mann Whitney test). 3.2. Apoptosis in T cells from peripheral blood and spleen To examine apoptosis in T cells from PBMC and spleen of L. chagasi-infected dogs, PBMC and spleen samples were evaluated immediately following collection. Apoptosis of T cells from PBMC and spleen was detected using commercial kits for both, Annexin V (Guava, Hayward, CA) and Caspases (Guava, Hayward, CA) and simultaneously anti-CD3 mAbs (Serotec, UK). itology 184 (2012) 147– 153 The percentage of apoptotic T cells was evaluated after CD3 positive cells were gated in Plot 1 (Fig. 2a), followed by detection double stained for apoptosis detection in cells gated in Plot 1 (Fig. 2b). The PBMC showed higher numbers of apoptotic T cells in infected dogs (Fig. 2c) compared to healthy dogs (Fig. 2d) and in the spleen, similar results were obtained (Fig. 2e and f). Data presented in Fig. 3 clearly indicate that T lympho- cytes from PBMC and spleen tissue of infected dogs showed a significantly higher level of apoptosis compared to that observed in healthy dogs (P < 0.05, Mann Whitney test). In general both kits used showed similar values of apoptotic cells in controls and infected dogs (Fig. 3). 3.3. White pulp structural organization and CD3 IHQ The percentage of dogs with slightly disorganized white pulp, with either hyperplastic or hypoplastic changes lead- ing to a loss of definition of any of the regions of the white pulp areas was 63.6%, while 36.3% dogs showed white pulp areas that were moderately or extensively disorganized and were poorly individualized or indistinct. The CD3 IHC confirmed the structural disorganization of the white pulp in infected dogs compared to that observed in healthy dogs (Fig. 4). A significant effect on the level of white pulp mor- phological disorganization and percentage of apoptosis in T cells from the spleen was observed (F = 20.45; P = 0.0014, least squares method). 4. Discussion The level of apoptosis in T cells from PBMC and spleen from VL dogs from an endemic area was analyzed and the values compared to uninfected control dogs to investigate the role this mechanism plays in the pathology and infec- tious process of the disease, as well as in the host immune response. Apoptosis is essential for homeostatic control of lym- phocyte numbers, particularly following the development of an immune response to an invasive microorganism (Scaffidi et al., 1999). However, some microbes are able to either directly manipulate this response or have devel- oped strategies to survive in the host until homeostatic mechanisms are activated to reduce effector T cell numbers (Freire-de-Lima et al., 2000). The apoptosis level in T cells from the spleen and periph- eral blood were higher in infected dogs compared to that of healthy canines, suggesting that the infection by L. (L.) cha- gasi could induce apoptosis in T cells. Since the progression of infection is related to the impairment of cell-mediated immunity (Dos Santos et al., 2008), the detection of T cell apoptosis may contribute to inefficient cell immune response during L. chagasi infection. Several mechanisms of T cell apoptosis have been pro- posed. Deprivation of growth factors, cytotoxicity by TNF-� and FAS and FAS-L interaction are among the major media- tors (Scaffidi et al., 1999). Leishmania spp. infection studies have shown that macrophages infected by L. donovani increased the level of FAS in the membrane and sFASL in the culture supernatant (Eidsmo et al., 2002) and in L. donovani infected Balb/c mice, low IL-2 levels in the T cell culture V.M.F.d. Lima et al. / Veterinary Parasitology 184 (2012) 147– 153 151 Fig. 2. Flow cytometry analysis: (a) fluorescence plot for gated lymphocytes from Leishmania infected dogs (Anti-CD3 monoclonal antibody FITC). Percentage inside upper right quadrants correspond to lymphocyte CD3 positive cells; (b) a representative fluorescence plot for gated lymphocytes and stained for apoptosis (positive control, the cells were incubated with apoptosis inductor Camptothecin); (c) a representative fluorescence plot for gated lymphocytes from PBMC from Leishmania infected dogs and stained for apoptosis; (d) a representative fluorescence plot for gated lymphocytes from PBMC from healthy dogs and stained for apoptosis; (e) a representative fluorescence plot for gated lymphocytes from the spleen of Leishmania-infected dogs and stained for apoptosis; and (f) a representative fluorescence plot for gated lymphocytes from the spleen of healthy dogs and stained for apoptosis. Percentage inside upper right quadrants correspond to lymphocytes stained with Annexin V-PE and 7-AAD fluorescence (double stained), late stage of apoptotic cells. Similarly dot plot figures were observed using the multicaspase kit. CT- Anexin V INF-Anexin V 0 5 10 15 P=0.009 Spleen % a po pt ot ic T ly m ph oc yt es CT-MulticaspaseINF-Multicaspase 0 10 20 P=0.001 Spleen % a po pt ot ic T ly m ph oc yt es CT-MulticaspaseINF-Multicaspase 0 5 10 15 P=0.001 Blood % a po pt ot ic T ly m ph oc yt es CT-Anexin VINF-Anexin V 0 5 10 15 20 P=0.0007 Blood % a po pt ot ic T ly m ph oc yt es Fig. 3. Apoptosis of T lymphocytes in infected dogs (INF) and control dogs (CT). Each circle represents an individual dog. Bars represent the mean value in each group. P < 0.05: significant differences between the mean values for Leishmania infected dogs and healthy controls. 152 V.M.F.d. Lima et al. / Veterinary Paras Fig. 4. Immunohistochemical analysis of CD3+ lymphocytes in the spleen of Leishmania infected dogs (a) and (b) and healthy control dogs (c): (a) the slight disorganization of the regions of white pulp; and (b) the exten- sive disorganization of the regions of white pulp. The lymphoid follicles with acute VL suggest that the dog is an excellent model showed a germinal center (GC) and a marginal zone (MZ). Avidin-biotin immunoperoxidase stained. In (a)–(c) the bars represent 200 �m. supernatant cause apoptosis of Th1-like cells (Das et al., 1999), the mechanism that induces apoptosis in T cells from dogs naturally infected with Leishmania spp. has not yet been established and remains to be defined. Few studies in the literature have investigated apopto- sis in trypanosomatid infection, though in myocarditis of experimental canine chagas disease, abundant apoptosis itology 184 (2012) 147– 153 of lymphocytes was observed (Zhang et al., 1999) simi- lar to the present results. The possibility that apoptosis may contribute to the pathogenesis and clinical status of leishmaniasis has recently been suggested. Leishmania and its membrane constituents have been shown to result in activation-induced apoptosis of CD4 T cells in vitro (Wolday et al., 1999). Similarly, in mice infected intravenously with L. donovani, significant T cell apoptosis was detected in spleen tissue compared to controls (Alexander et al., 2001). Lymphoid disorganization in the white pulp in spleen was present in the infected dogs examined in this study and cachexia was frequently observed, similar to that observed by Santana et al. (2008), who reported loss of lymphoid follicle definition in underweight dogs. Tissue lymphoid disorganization is not only related with cachexia, TNF-� is also related with wasting in visceral leishmaniasis (Pearson et al., 1990) and the TNF-� production mediates loss of the architectural structure of spleen tissue in murine models of visceral leishmaniasis (Engwerda et al., 2002). Alike mice, in dogs naturally infected by Leishmania spp. high levels of TNF-� are produced by spleen cells indicating that the presence of L. (L.) chagasi induces an immune response with relevant expression of this cytokine (Michelin et al., 2011). TNF-� is also involved in apoptosis mechanisms (Kanaly et al., 1999). The relation observed between a high percent- age of apoptosis and the structural disorganization of white pulp suggests that apoptosis is involved in lymphoid tissue disorganization and the role of TNF-� in both processes should be clarified in the future. The higher levels of apoptosis observed in T cells from the spleen and PBMC of infected dogs appear to be a phys- iological response to persistent immune activation; the mechanisms involved have yet to be studied. The deple- tion of T cells probably reflects the low T cell immunity response verified in symptomatic dogs that presented high parasitism in the spleen (Sanchez et al., 2004). The partic- ipation of T lymphocytes in the granuloma formation to control Leishmania sp. infection has been shown (Murray, 2001). In infected symptomatic dogs, the lack of mature and well organizated granulomas in the spleen (Sanchez et al., 2004) could be related to apoptosis, the infiltrated T cells in the spleen of symptomatic dogs represent a nonstructural nonfunctional granuloma, such as those observed in T cell deficient mice (Murray, 2001). It is possible that apoptotic mechanisms may eventually develop into nonspecific T cell depletion, making dogs with VL more susceptible to other infections, especially ehrli- chiosis and babesiosis. Such coinfections are frequently observed in VL symptomatic dogs (Oliveira et al., 2008) and, occasionally, neoplastic disease, especially hematopoietic tumors, are involved (Foglia Manzillo et al., 2008). Observation of apoptosis in T lymphocytes from symp- tomatic naturally infected dogs, confirming studies in patients with acute visceral leishmaniais where CD4+ T cells from PBMC undergo significant levels of apoptosis (Potestio et al., 2004). Similarities observed in immunolog- ical response between naturally infected dogs and patients for studying new therapies. Taken together, these data indicate that in infected dogs, the immunosuppression associated with chronic infection ry Paras i a t T u w t i e A f R A A B C C D D D D E E F F F F G K V.M.F.d. Lima et al. / Veterina s due to accelerated rates of T cell apoptosis and this mech- nism could contribute to white pulp disorganization in he spleen and diminished T cell levels in peripheral blood. he present results could contribute to improving current nderstanding of the immune response in dogs infected ith L. (L.) chagasi, while additional studies would fur- her our understanding concerning apoptosis and other mmune mediators in dogs naturally infected with this dis- ase. cknowledgements The authors are grateful to the FAPESP and FUNDUNESP or financially supporting this project. eferences lexander, C.E., Kaye, P.M., Engwerda, C.R., 2001. 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(L.) chagasi naturally infected dogs 1 Introduction 2 Materials and methods 2.1 The study area 2.2 Ethical issue 2.3 Animals and diagnosis of VL 2.4 Sample collection 2.5 Kalazar detect rapid test rK-39 (InBios International, USA) 2.6 PCR 2.7 Apoptosis evaluation in T cells 2.8 Spleen samples and histological analysis 2.9 Immunohistochemistry (IHC) 2.10 Statistical analysis 3 Results 3.1 Decrease in T lymphocytes in PBMC 3.2 Apoptosis in T cells from peripheral blood and spleen 3.3 White pulp structural organization and CD3 IHQ 4 Discussion Acknowledgements References