Early-life longitudinal survey of peripheral blood lymphocyte subsets in Beagle dogs

A longitudinal study of peripheral blood lymphocyte subsets from 7 to 15 months of age was performed in Beagle dogs employing a multiparametric flow cytometry. The data were compared with data obtained from adult Beagle dogs that were housed in the same animal facilities and that were subjected to the same controls during the 34 weeks of the study. Absolute counts of total lymphocytes and CD3+ T, CD3+CD4+ Th and CD21+ B lymphocytes decreased during the entire 34 weeks period of the study in the young dogs group. The same was observed with regard to the percentage of CD3+CD4+ Th lymphocytes and the CD4/CD8 ratio, while the percentage of CD3+CD8+ Tc lymphocytes increased from 7 to 15 months of age. These age-related changes found in lymphocyte subsets distribution of young dogs led to level the absolute and relative values of adult dog lymphocytes. The observations of this longitudinal study illustrate the changes related to maturation of lymphocyte subsets that occur during early life in Beagle dogs.     

鸡毒支原体F弱毒疫苗株细胞免疫特性研究

为了解鸡毒支原体（Mycoplasma gallisepticum,MG）F弱毒疫苗株的细胞免疫特性,分别以活菌浓度为109（A组）、106（B组）ccu/mL的MG F株及生理盐水（C组）点眼接种SPF鸡,采用流式细胞技术及T淋巴细胞增殖试验对免疫前后淋巴细胞亚类Th/T、Tc/T、Th/Tc及刺激指数（SI）的动态变化规律进行研究。结果显示免疫后A、B组的Th/T、Tc/T、SI明显升高,其中Th/T于d5、d7,Tc/T、SI于d7、d14,A组显著高于B组（P〈0.05）。研究结果表明,免疫MG F株可较好的提高鸡的细胞免疫,且MG F株活菌浓度与接种鸡外周血Th/T、Tc/T、SI呈正相关。     

猪圆环病毒2型感染对伪狂犬疫苗免疫应答的影响

为明确PCV2感染对伪狂犬(PR)疫苗免疫应答的影响,本研究采用阻断ELISA方法对单独接种猪PR疫苗组(A组)及PCV2人工感染3周后接种猪PR疫苗组(PA组)不同时相血清中的猪PR病毒gB抗体进行检测;同时对不同时相前腔静脉血进行CD4+/CD8+流式细胞术及血常规分析。结果表明,在PCV2感染后2周至5周间,A组白细胞含量均高于PA组,随后PA组白细胞恢复至与A组略高的正常水平;在整个实验中,除接种猪PR疫苗后1周(WPI)和9周(WPI)外的所有时相PA组的淋巴细胞含量均略高于A组;PCV2感染后可使记忆/激活Th细胞数量略有升高,幼稚型Th细胞含量的下降;PCV2感染后2周～7周PA组Tc细胞均高于A组,在9WPIPA组Tc细胞数量显著下降(p0.05);除9WPI外,A组的S/N值均低于PA组。结果表明,PCV2感染看降低机体产生针对PRVgB特异性抗体水平,而且在一定程度上降低了幼稚型Th细胞及Tc细胞含量。     

Cytologic patterns of lymphadenopathy in canine monocytic ehrlichiosis

Background: Recognition of different cytologic patterns in lymph nodes (LNs) from dogs with canine monocytic ehrlichiosis (CME) and noninfectious causes of lymphoid reactivity may have diagnostic utility. Objectives: The aims of the present study were to compare cytologic patterns in LNs of dogs with different phases of CME, to investigate the association of cytologic pattern and presence of Ehrlichia spp. morulae, and to compare patterns of lymphoid reactivity between dogs with CME and those with noninfectious causes of lymphoid hyperplasia. Methods: Cytologic preparations of LNs from 35 dogs with nonmyelosuppressive CME (group A), 16 dogs with myelosuppressive CME (group B), 26 dogs with noninfectious diseases (group C), and 15 healthy dogs (group D) were evaluated. Percentages of lymphocyte types, plasma cells, macrophages, neutrophils, and eosinophils were determined. Samples from dogs in groups A and B were evaluated for the presence of morulae. Results: Cytologic abnormalities in LNs were recorded in 54% of dogs in group A, 88% in group B, 39% in group C, and 0% in group D and were more frequent (P=.02) in dogs with myelosuppressive CME than those with nonmyelosuppressive CME. Plasma cell hyperplasia was more frequent in CME than in noninfectious diseases (P=.03). An association between the presence of cytologic abnormalities and morulae in group A dogs was not found. Conclusions: Dogs with myelosuppressive CME have more lymphoid cytologic abnormalities than dogs with nonmyelosuppressive CME. LN plasmacytosis is the major pattern of lymphadenopathy in dogs with CME and is found more frequently in dogs with CME than in dogs with noninfectious causes of lymphadenopathy.     

Serum acute phase proteins as clinical phase indicators and outcome predictors in naturally occurring canine monocytic ehrlichiosis

Background: Canine monocytic ehrlichiosis (CME), caused by Ehrlichia canis, is an important tick‐borne disease of global importance. Currently, limited information is available on the diagnostic and prognostic value of acute phase proteins (APPs) in dogs naturally infected with E. canis. Hypothesis: APPs may be useful indicators of the clinical phase of CME and predictive of the clinical outcome (death or survival). Animals: Fifty‐six dogs naturally infected with E. canis and 7 clinically healthy control dogs. Methods: C‐reactive protein (CRP), serum amyloid A (SAA), haptoglobin (Hp), and albumin concentrations determined on admission were retrospectively compared among 27 dogs with nonmyelosuppressive CME, 29 dogs with myelosuppressive CME and 7 healthy dogs. Diagnosis of CME was based on clinical and clinicopathological findings, seropositivity to E. canis, polymerase chain reaction amplification of E. canis‐specific 16S rDNA, microscopic observation of Ehrlichia sp. morulae in blood monocytes or some combination of these. Results: Mean concentrations of CRP, SAA, and Hp were significantly higher in the myelosuppressed dogs compared with the other groups, but no significant differences were found in the concentration of albumin. Survival analysis of the affected animals indicated that APP concentrations were not associated with clinical outcome; the latter was strongly associated with pancytopenia (odds ratio for death 22.7) and neutropenia (odds ratio for death 7.7). Conclusions and Clinical Importance: CRP, SAA, and Hp serum concentrations on admission are useful indicators of the clinical phase of CME, but are not useful predictors of clinical outcome.     

An immunologic investigation of canine eosinophilic bronchopneumopathy

Immunologic variables in dogs with eosinophilic bronchopneumopathy (EBP) have not been extensively evaluated. The aim of this study was to determine immunoglobulin (Ig) concentrations and to perform phenotypic subtyping of lymphocytes in the bronchoalveolar lavage fluid (BALF) and peripheral blood (PB) of 12 dogs with EBP at the time of diagnosis (TD) and to compare these data with those obtained in healthy dogs, as well as in EBP dogs after antibiotic therapy (TAB) and during corticosteroid treatment (TM). Matched samples of serum and BALF were used to determine Ig concentrations (IgG, IgM, and IgA) by capture enzyme-linked immunosorbent assay (ELISA), from which a secretory index (SI) was calculated. Lymphocyte subpopulations were studied in the BALF and PB by flow cytometry. Log values of BALF IgM and IgA were significantly higher (0.64+/-0.05 and 1.06+/-0.13, respectively) in EBP dogs at TD than in controls and then tended to decrease at TM (0.55+/-0.03 and 1.02+/-0.17, respectively). A calculated SI for IgA was not significantly increased. In the BALF of dogs with EBg the CD4: CD8 was significantly (P < .05) higher (22.6+/-30.3) than in controls (3.2+/-1.9), due to significantly higher CD4+ T cells and lower CD8+ T cells. At TM, the BALF T-cell percentages returned to normal (2.4+/-0.6). We propose that the influx of eosinophils into the airway of dogs with EBP is at least in part mediated by cytokines derived from CD4+ T cells. Further studies of canine cytokines and chemokines will help determine whether canine EBP involves type I hypersensitivity mechanisms regulated by Th2 lymphocytes.     

Flow cytometric assay in peripheral blood of dogs--reference values for leukocytes from Brazilian beagles

Use of domestic reference values in the flow cytometry analysis is known to improve its accuracy by integrating local variations as gender, race and age. Up to date application of flow cytometry in veterinary medicine has been limited to describe the percentual values just for peripheral lymphocytes subsets of blood. We now report establishment of reference values for a wide range of proportional and absolute numbers of peripheral blood leukocytes, including T cells subsets, B cells, monocytes and eosinophils, applicable to the healthy population of Beagles in Brazil and other regions with similar demographic characteristics. Normal reference values were also established to estimate the gender-related differences. This information will provide clinical aid in the evaluation of immunologic status as well as standard values for experimental animals of dogs from Brazil and other similar regions.     

Alterations of lymphocyte subpopulations in healthy dogs with aging and in dogs with cancer

Changes in an individual's immune status are considered major contributing factors towards the morbidity of cancer and mortality of aging. To evaluate age-related changes in the immune status of dogs, the immunophenotypes (CD3, CD4, CD8 and CD21) of peripheral blood lymphocytes were measured in 160 healthy dogs aged from 1 to 17 years, and in 365 dogs with various tumors and at various stages. In healthy dogs, the absolute numbers of white blood cells, lymphocytes, and CD3(+), CD4(+) and CD21(+) lymphocytes decreased significantly with age. The relative percentages of lymphocytes and CD4(+) cells decreased significantly, while CD8(+) cells increased significantly with age. The CD4:CD8 ratio showed a significant age-related decrease. In contrast, dogs with tumors possessed significantly lower absolute numbers and relative percentages of all lymphocyte phenotypes, while the CD4:CD8 ratio was significantly higher than in age-matched controls. The relative percentages of CD3(+) and CD8(+) lymphocytes were significantly lower in dogs with distant metastases compared with dogs without metastases, and the CD4:CD8 ratio increased with advanced stage. These observations illustrate the significant changes in immune status with age and the presence of marked immunological defects in a large-scale study of dogs with advanced tumors.     

Bone marrow mastocytosis in dogs with myelosuppressive monocytic ehrlichiosis (Ehrlichia canis): a retrospective study

BACKGROUND: Bone marrow mastocytosis has been reported rarely in naturally occurring canine monocytic ehrlichiosis (CME). OBJECTIVES: The aims of the present study were to estimate the prevalence and magnitude of bone marrow mastocytosis in a case series of dogs with natural CME and to assess the association, if any, between mastocytosis and the clinical severity of the disease. METHODS: Seventy-six dogs with confirmed CME (Ehrlichia canis) were included in the study. Affected dogs were allocated into group A (n = 51) without bone marrow hypoplasia and group B (n = 25) with bone marrow hypoplasia. Twenty clinically healthy Beagles not previously exposed to E canis served as controls (group C). The main inclusion criteria for group A were documentation of normocellular to hypercellular bone marrow and complete clinical cure following a 4-week treatment with doxycycline, while those for group B were bone marrow hypoplasia and lack of response to doxycycline. Bone marrow aspirate smears from all 96 dogs were Giemsa-stained and examined for the presence of mast cells, which were calculated as a percentage of 1,000 nucleated cells (NCs). The prevalence of mastocytosis was compared among the 3 groups by the Pearson's chi-square test. RESULTS: Bone marrow mastocytosis (>0.1% of NCs) was found in 5 (20%) dogs in group B (range, 0.5-2.5% of NCs; median, 1% of NCs). One dog in each of groups A and C had 0.1% mast cells in the marrow. The prevalence of bone marrow mastocytosis in dogs in group B was significantly higher (P = .004) than in groups A and C. CONCLUSION: Bone marrow mastocytosis can be seen in a substantial number of dogs with E canis-induced myelosuppression.     

In vitro mitogen responses and lymphocyte subpopulations in cheetahs.

Lack of genetic variability and apparent susceptibility of cheetahs (Acinonyx jubatus jubatus) to coronavirus infection has lead to speculation that this species may have immune system deficits. To establish a foundation for evaluation of the immune function, cheetah peripheral blood mononuclear cells (PBM) were stimulated by a panel of six mitogens, and responses compared with those of domestic cat PBM. Individual responses in both species were variable, but evenly distributed throughout the range of stimulation for each mitogen. Proliferation by PBM from domestic cats occurred within the same range as that of the cheetahs. However, a significantly lower response to peanut agglutinin (PNA) was observed with domestic cat PBM. Although responses varied between animals, certain individual cheetahs were consistent low responders. The decreased values could not be explained by lack of IL-2 responsiveness since exogenous IL-2 significantly enhanced mitogen-stimulated proliferation in 11 of 12 cheetahs tested. The phenotypic distribution of domestic cat and cheetah lymphocyte subpopulations was similar as assessed by immunofluorescence staining for surface immunoglobulin (sIg) and cytotoxic T (Tc) cells (using a specific monoclonal antibody, FT2). Values for B cells (31.2% sIg+) and Tc (28.7% FT2+) were slightly higher in domestic cats as compared with cheetah PBM (13.3% sIg+; 19.0% FT2+). Even though no species-specific deficits were detected, a significant negative correlation between PHA-stimulated proliferation and percent FT2+ (Tc) cheetah cells was observed. This indicates that proliferation can be used indirectly to assess relative numbers of functional T helper cells in cheetahs. Our studies suggest that these aspects of the cheetah's immune system are comparable with the domestic cat, and establish a basis for in vitro assays evaluating antigen-specific responses.     

Evaluation of a serum-based PCR assay for the diagnosis of canine monocytic ehrlichiosis

The aim of this study was to estimate the relative diagnostic sensitivity and specificity of a polymerase chain reaction (PCR) assay in the serum of dogs with naturally occurring non-myelosuppressive canine monocytic ehrlichiosis (CME), and to investigate the association between PCR positivity and immunofluorescence antibody (IFA) titres for Ehrlichia canis. Serum samples obtained from 38 dogs with non-myelosuppressive CME and 12 healthy dogs were analyzed retrospectively. Each serum sample was analyzed in triplicate using an E. canis-specific nested PCR assay targeting a 389 bp sequence of the 16S rRNA gene. E. canis DNA was amplified in 24 of 38 (63.1%) affected dogs; all samples from healthy dogs were negative. A high level of agreement was found among the PCR replicates (P < 0.0001). Median IFA titre of the 24 PCR-positive dogs was significantly lower than that of the PCR-negative infected dogs (P = 0.0029), indicating that E. canis DNA may circulate prior to the development of a high antibody titre. Serum-based PCR analysis is suggested for the early diagnosis of CME when whole blood samples are not available.     

Measurement of thrombopoietic activity through the quantification of megakaryocytes in bone marrow cytology and reticulated platelets

Reticulated platelets are considered as marker for bone marrow thrombopoiesis. The aim of the study was evaluate the role of reticulated platelets as markers of thrombopoiesis in dogs. Reticulated platelets analysis by flow cytometry and megakaryocyte quantification by bone marrow cytology were determined in 29 healthy adult dogs (control group), 14 dogs with thrombocytopenia without megakaryocytic hypoplasia (group A) and 14 dogs with thrombocytopenia which presented megakaryocytic hypoplasia (group B), detected by bone marrow aspiration cytology. Blood samples were collected and the platelet rich plasma was obtained for reticulated platelets quantification in flow cytometry. Megakaryocytes were quantified in aspiration cytology by two techniques in marrow particles, and correlated to reticulated platelets counts. There are no differences between megakaryocyte quantification. Although there is no correlation between reticulated platelet values and megakaryocyte in bone marrow cytology, the interpretation of reticulated platelet values can be based both on absolute or relative corrected values.     

Immunopathology of Bartonella vinsonii (berkhoffii) in experimentally infected dogs.

Following natural infection with Bartonella, dogs and humans develop comparable disease manifestations including endocarditis, peliosis hepatis, and granulomatous disease. As the immunologic response to infection in these hosts has not been clearly established, data presented here was derived from the experimental infection of six specific pathogen free (SPF) beagles with a known pathogenic strain of Bartonella. Six dogs were inoculated intravenously with 10(9)cfu of B. vinsonii ssp. berkhoffii and six control dogs were injected intravenously with an equivalent volume of sterile saline. Despite production of substantial levels of specific antibody, blood culture and molecular analyses indicated that Bartonella established chronic infection in these dogs. Flow cytometric analysis of monocytes indicated impaired bacterial phagocytosis during chronic Bartonella infection. There was also a sustained decrease in the percentage of CD8+ lymphocytes in the peripheral blood. Moreover, modulation of adhesion molecule expression (downregulation of L-selectin, VLA-4, and LFA-1) on CD8+ lymphocytes suggested quantitative and qualitative impairment of this cell subset in Bartonella-infected dogs. When compared with control dogs, flow cytometric analysis of lymph node (LN) cells from B. vinsonii infected dogs revealed an expanded population of CD4+ T cells with an apparent na?ve phenotype (CD45RA+/CD62L+/CD49D(dim)). However, fewer B cells from infected dogs expressed cell-surface MHC II, implicating impaired antigen presentation to helper T cells within LN. Taken together, results from this study indicate that B. vinsonii establishes chronic infection in dogs which may result in immune suppression characterized by defects in monocytic phagocytosis, an impaired subset of CD8+ T lymphocytes, and impaired antigen presentation within LN.     

Frequent respiratory tract infections in the canine model of X-linked ectodermal dysplasia are not caused by an immune deficiency

As in many human patients with X-linked hypohidrotic ectodermal dysplasia (XHED), XHED dogs are at an increased risk for pulmonary disorders. Localized immune system defects had been suspected previously in affected dogs because of frequent infections and unexpected deaths due to opportunistic respiratory tract infections. Experiments were designed to examine systemic and localized humoral and cellular responses, development and function of T cells, and thymic morphology. All dogs used in these experiments were clinically healthy at the time of examination and their immune responses were compared to normal littermates. Serum immunoglobulin concentrations differed somewhat between normal dogs and dogs affected with XHED but they were all within normal ranges. The XHED dogs responded appropriately to vaccination with tetanus toxoid suggesting normal systemic B and plasma cell function. Thymic morphology was compared between normal and affected dogs and T cells were assessed for functionality. Numbers and phenotypes of T and B cells in blood and thymus of affected dogs were within normal limits suggesting normal development of T cells. Cytotoxic and phagocytic ability of macrophages and neutrophils was also normal in affected dogs. In contrast, the secretory IgA concentrations found in affected dogs were significantly higher than in normal dogs, while lacrimal secretions were significantly decreased. These results suggest a compensatory mechanism for secretory IgA, so that the total amount equals that in normal dogs. The results presented in this study indicate that the XHED dogs have a relatively intact immune system and suggest that the same is true for humans with the homologous form of XHED.     

Evaluation of neutrophil oxidative metabolism in canine monocytic ehrlichiosis

BACKGROUND: Canine monocytic ehrlichiosis (CME) is a tick-borne disease caused by Ehrlichia canis, a rickettsia that infects the monocytes of dogs. This infection can result in a chronic and life-threatening disease. Thrombocytopenia, mild anemia, and leukopenia are the most common hematologic findings in CME. OBJECTIVE: To investigate the role of peripheral blood neutrophils in CME, an evaluation was conducted of their functional state during the acute phase of the disease in dogs experimentally infected by E canis. METHODS: Seven dogs were inoculated with E canis, and 3 remained as uninfected controls. All dogs had physical exams and hematologic tests (CBC and nitroblue tetrazolium [NBT] reduction) during a 6-week period. RESULTS: There was no difference (P > .05) in spontaneous NBT reduction results between the 2 groups of dogs throughout the 6-week period of observation. Nevertheless, when stimulated, the neutrophils showed higher activity in the infected group (P = .01) on weeks 4 and 5 after infection. CONCLUSION: Infection by E canis has no influence on neutrophil oxidative metabolism even though during the remission period of the acute phase of the disease, the neutrophils seem to be more reactive under stimulation.     

Humoral and cellular immune responses in dogs with inapparent natural Leishmania infantum infection

Molecular analysis, serology and immunophenotyping for T lymphocytes and their subsets, B lymphocytes and monocytes were performed on dogs naturally infected with Leishmania infantum. Animals were categorised as asymptomatic dogs I (AD-I), with negative serology and positive molecular results, and asymptomatic dogs II (AD-II), with positive serology and positive molecular results, and these were compared to symptomatic dogs (SD) and control dogs (CD). AD-I exhibited immunophenotypic features similar to those of CD, including isotype profiles and concentrations of monocytes. Similar biomarkers were found in AD-II and SD, such as, higher levels of immunoglobulins IgG, IgG2, IgM and IgA and higher concentrations of eosinophils. High frequencies of T lymphocytes and CD4(+) T cells were observed in both AD-I and AD-II compared to SD, whereas CD8(+) T cells were higher only in AD-II compared with SD. Analysis of B lymphocytes revealed an increased frequency of this cell type only in AD-II animals compared with SD. Asymptomatic dogs appear to have a dichotomous infection spectrum that can influence the humoral and cellular immunological status during canine visceral leishmaniasis.     

Experimental Ehrlichia canis infection in the dog does not cause immunosuppression

A carrier state develops in some Ehrlichia canis-infected dogs due to ineffective host defenses. The subsequent development of immune-mediated diseases or opportunistic infections in chronic ehrlichiosis suggests dysregulation of immunity; however, the immunobiology of this infection has not been well characterized. In this study, eight dogs were infected with E. canis, and changes in seroreactivity, serum immunoglobulin (Ig) concentrations, peripheral blood T cell subsets, lymphocyte blastogenesis (LBT), and lymphokine-activated killer (LAK) activity were evaluated over 4 months. Infection, which was documented by seroconversion, polymerase chain reaction, and blood culture, caused self-limiting fever and thrombocytopenia. Infected dogs developed an anti-E. canis antibody response but were not immune to re-infection. Serum IgM, IgG, and IgA concentrations were unaffected by E. canis. The percentage of circulating CD4(+) T cells was similar in uninfected and infected dogs at all points. Infected dogs developed a CD8(+) lymphocytosis 6 weeks after inoculation that subsequently subsided, despite organism persistence. Functional defects of cell-mediated immunity, measured as suppression of LAK activity or mitogen-driven LBT, were not observed. These results suggest that immune responses are not grossly impaired in young dogs during the first several months following experimental E. canis infection.     

Peripheral Blood Lymphocyte Subpopulations and Immunoglobulin Concentrations in Healthy Foals and Foals with Rhodococcus equi Pneumonia

Infectious diseases are common in foals aged 1-5 months. The objectives of this investigation were to evaluate immunologic parameters in foals from birth to weaning to establish reference values for the proportion of circulating lymphocytes that were helper (CD4+) or cytotoxic (CD8+) T cells, or B cells; to measure serum immunoglobulin (IgM and IgG) concentrations; and to compare these immunologic parameters to values in foals with naturally occurring Rhodococcus equi pneumonia and in adult horses. Peripheral blood lymphocyte subpopulations were determined by flow cytometric analysis, and serum IgG and IgM concentrations were determined by radial immunodiffusion. Flow cytometric analysis of lymphocyte subpopulations suggested age-related changes in the cell-mediated immune system in horses. Absolute circulating CD4+ and CD8+ T lymphocytes and B cells increased linearly up to 3 months of age. Circulating B cell concentrations from birth to 6 months of age were greater than values in adult horses and the lymphocyte differences among the age groups are mainly due to variation in B lymphocytes. Both absolute and proportional B cell concentrations were greater in foals with R equi pneumonia than in healthy foals at the same age. The increase in absolute cell counts of each subpopulation was dependent on the increase of absolute peripheral blood lymphocyte count. Serum IgG concentration increased linearly from 1 to 3 months of age, and serum IgM concentrations increased from 1 to 6 months of age. These data suggest age-dependent cell-mediated and humoral development in young foals.     

Apoptosis in T lymphocytes from spleen tissue and peripheral blood of L. (L.) chagasi naturally infected dogs

Dogs are the main domestic reservoirs of L. (L.) chagasi. Once in the vertebrate host, the parasite 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 symptomatic 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 Multicaspase 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). Apoptosis 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.