Risk factors for gastrointestinal parasite infections of dogs living around protected areas of the Atlantic Forest: implications for human and wildlife health / Fatores de risco para infecções parasitárias gastrointestinais em cães do entorno de áreas protegidas da Mata Atlântica: implicações para a saúde humana e da vida selvagem

Abstract Despite the ubiquity of domestic dogs, their role as zoonotic reservoirs and the large number of studies concerning parasites in urban dogs, rural areas in Brazil, especially those at the wildlife-domestic animal-human interface, have received little attention from scientists and public health managers. This paper reports a cross-sectional epidemiological survey of gastrointestinal parasites of rural dogs living in farms around Atlantic Forest fragments. Through standard parasitological methods (flotation and sedimentation), 13 parasite taxa (11 helminths and two protozoans) were found in feces samples from dogs. The most prevalent were the nematode Ancylostoma (47%) followed by Toxocara (18%) and Trichuris (8%). Other less prevalent (<2%) parasites found were Capillaria, Ascaridia, Spirocerca, Taeniidae, Acantocephala, Ascaris, Dipylidium caninum, Toxascaris, and the protozoans Cystoisospora and Eimeria. Mixed infections were found in 36% of samples, mostly by Ancylostoma and Toxocara. Previous deworming had no association with infections, meaning that this preventive measure is being incorrectly performed by owners. Regarding risk factors, dogs younger than one year were more likely to be infected with Toxocara, and purebred dogs with Trichuris. The number of cats in the households was positively associated with Trichuris infection, while male dogs and low body scores were associated with mixed infections. The lack of associations with dog free-ranging behavior and access to forest or villages indicates that infections are mostly acquired around the households. The results highlight the risk of zoonotic and wildlife parasite infections from dogs and the need for monitoring and controlling parasites of domestic animals in human-wildlife interface areas.

Resumo Apesar da ubiquidade dos cães domésticos, de seu papel como reservatório de doenças, e do grande número de estudos sobre parasitas de cães urbanos, as áreas rurais no Brasil, especialmente aquelas na interface entre animais silvestres - animais domésticos - humanos, tem recebido pouca atenção de cientistas e gestores de saúde pública. Este artigo relata um estudo epidemiológico seccional de parasitas gastrointestinais de cães rurais em propriedades no entorno de fragmentos de Mata Atlântica. Através de métodos parasitológicos como flutuação e sedimentação, 13 táxons de parasitas (11 helmintos e dois protozoários) foram encontrados em amostras de fezes dos cães. O mais prevalente foi o nematóide Ancylostoma (47%), seguido por Toxocara (18%) e Trichuris (8%). Outros parasitas menos prevalentes (<2%) encontrados foram Capillaria, Ascaridia, Spirocerca, Taeniidae, Acantocephala, Ascaris, Dipylidium caninum, Toxascaris, e os protozoários Cystoisospora and Eimeria. Infecções mistas foram detectadas em 36% das amostras, a maioria por Ancylostoma e Toxocara. Vermifugações prévias não foram associadas a infecções, indicando que esta medida preventiva está sendo realizada incorretamente pelos proprietários. Com relação aos fatores de risco, cães com menos de um ano tiveram maior probabilidade de infecção por Toxocara, e os cães de raça pura por Trichuris. O número de gatos na propriedade foi associado positivamente com a infecção por Trichuris, enquanto cães machos e baixos escores corporais foram associados a infecções mistas. A ausência de associações com comportamento de vida livre e acesso a florestas ou vilas pelos cães indica que as infecções estão sendo predominantemente adquiridas nas propriedades. Os resultados destacam o risco de infecções parasitárias zoonóticas e para animais silvestres a partir dos cães, e a necessidade de monitorar e controlar os parasitas de animais domésticos em áreas de interface entre humanos e a vida selvagem.

Effects of miRNA-15 and miRNA-16 expression replacement in chronic lymphocytic leukemia: implication for therapy.

Chronic lymphocytic leukemia (CLL) clones are characterized by loss of a critical region in 13q14.3, (del(13)(q14)) involving the microRNA (miRNA) cluster miR-15a and miR-16-1. We have investigated the effects of replacement of miR-15a and miR-16-1. CLL cells transfected with these miRNA mimics exhibited a decrease in cell viability in vitro and impaired capacity for engraftment and growth in NOD/Shi-scid,γcnull (NSG) mice. No synergistic effects were observed when the two miRNA mimics were combined. The phenomena were not restricted to CLL with the del(13)(q14) lesion. Similar effects induced by miRNA mimics were seen in cells with additional chromosomal abnormalities with the exception of certain CLL clones harboring TP53 alterations. Administration of miRNA mimics to NSG mice previously engrafted with CLL clones resulted in substantial tumor regression. CLL cell transfection with miR-15a and miR-16-1-specific inhibitors resulted in increased cell viability in vitro and in an enhanced capacity of the engrafted cells to grow in NSG mice generating larger splenic nodules. These data demonstrate that the strong control by miR-15a and miR-16-1 on CLL clonal expansion is exerted also at the level of full-blown leukemia and provide indications for a miRNA-based therapeutic strategy.

Risk factors for gastrointestinal parasite infections of dogs living around protected areas of the Atlantic Forest: implications for human and wildlife health.

Despite the ubiquity of domestic dogs, their role as zoonotic reservoirs and the large number of studies concerning parasites in urban dogs, rural areas in Brazil, especially those at the wildlife-domestic animal-human interface, have received little attention from scientists and public health managers. This paper reports a cross-sectional epidemiological survey of gastrointestinal parasites of rural dogs living in farms around Atlantic Forest fragments. Through standard parasitological methods (flotation and sedimentation), 13 parasite taxa (11 helminths and two protozoans) were found in feces samples from dogs. The most prevalent were the nematode Ancylostoma (47%) followed by Toxocara (18%) and Trichuris (8%). Other less prevalent (<2%) parasites found were Capillaria, Ascaridia, Spirocerca, Taeniidae, Acantocephala, Ascaris, Dipylidium caninum, Toxascaris, and the protozoans Cystoisospora and Eimeria. Mixed infections were found in 36% of samples, mostly by Ancylostoma and Toxocara. Previous deworming had no association with infections, meaning that this preventive measure is being incorrectly performed by owners. Regarding risk factors, dogs younger than one year were more likely to be infected with Toxocara, and purebred dogs with Trichuris. The number of cats in the households was positively associated with Trichuris infection, while male dogs and low body scores were associated with mixed infections. The lack of associations with dog free-ranging behavior and access to forest or villages indicates that infections are mostly acquired around the households. The results highlight the risk of zoonotic and wildlife parasite infections from dogs and the need for monitoring and controlling parasites of domestic animals in human-wildlife interface areas.

Gastrointestinal parasites of maned wolf (Chrysocyon brachyurus, Illiger 1815) in a suburban area in southeastern Brazil.

We examined 42 maned wolf scats in an unprotected and disturbed area of Cerrado in southeastern Brazil. We identified six helminth endoparasite taxa, being Phylum Acantocephala and Family Trichuridae the most prevalent. The high prevalence of the Family Ancylostomatidae indicates a possible transmission via domestic dogs, which are abundant in the study area. Nevertheless, our results indicate that the endoparasite species found are not different from those observed in protected or least disturbed areas, suggesting a high resilience of maned wolf and their parasites to human impacts, or a common scenario of disease transmission from domestic dogs to wild canid whether in protected or unprotected areas of southeastern Brazil.

High consumption of primates by pumas and ocelots in a remnant of the Brazilian Atlantic Forest.

We studied the diet of the ocelot and puma during the years 2007 and 2008 at the Feliciano Miguel Abdala Reserve, in Minas Gerais, south-eastern Brazil. We collected 49 faecal samples (scats) from cats, and identified the species of cat from 23 of them by the analysis of the microstructure patterns of hairs found in their faeces: 17 scats of the puma (Puma concolor) and six of the ocelot (Leopardus pardalis). In the puma scats, we identified three species of primates (Brachyteles hypoxanthus, Alouatta guariba and Sapajus nigritus), the remains of which were found in eight of 17 collected (47.1%), representing 26.7% of items consumed. For the ocelot, we detected capuchin monkey (S. nigritus) remains in three of the six scats (50%), accounting for 18.7% of items consumed by ocelot. We were unable to identify the cat species in the remaining 26 faecal samples, but we were able to analyse the food items present. Primates were found in five of these 26 faeces (19.2%) and represented 10.2% of the items found. Although the sample size is limited, our results indicate a relatively high consumption of primates by felines. We believe that this high predation may be the result of the high local density of primates as well as the greater exposure to the risks of predation in fragmented landscapes, which tends to increase the incidence of the primates using the ground.

Benznidazole treatment during early-indeterminate Chagas' disease shifted the cytokine expression by innate and adaptive immunity cells toward a type 1-modulated immune profile.

Trypanosoma cruzi-infected children was treated with benznidazole (Bz) during the early-indeterminate disease (E-IND) and the cytokine pattern of innate and adaptive immune compartments were evaluated prior to the treatment and 1 year after it. At first, we observed that the ex vivo cytokine profile of circulating leukocytes from E-IND (n = 6) resembled the one observed for healthy schoolchildren (n = 7). Additionally, in vitro stimulation with T. cruzi antigens drove the E-IND cytokine pattern toward a mixed immune profile with higher levels of IFN-gamma+, TNF-alpha+ and IL-4+ NK cells, increased numbers of IFN-gamma+, TNF-alpha+ and IL-10+ CD4+ T cells in addition to enhanced frequency of TNF-alpha+/IL-4+ CD19+ lymphocytes. Interestingly, upon T. cruzi antigen in vitro stimulation, E-IND CD8+ lymphocytes displayed a selective enhancement of IFN-gamma expression, accounting for a global type 1-modulated cytokine microenvironment. A shift toward a type 1-modulated profile was also the hallmark of Bz-treated children (E-IND(T)). In this context, despite the mixed overall ex vivo cytokine profile observed for NK and CD8+ T cells, increased ability of these leukocytes to produce IFN-gamma in response to T. cruzi antigens was reported. Most noteworthy was the IL-10 production evidenced at T lymphocytes, mainly CD4+ cells, as well as B lymphocytes, both ex vivo and upon antigen stimulation. Together, these findings gave evidence that NK cells and CD8+ T lymphocytes are the major sources of IFN-gamma, a pivotal cytokine for successful therapeutic response in human Chagas' disease. Moreover, our data have also brought additional information, pointing out IL-10 production by CD4+ cells and B lymphocytes, as the putative key element for parasite clearance in the absence of deleterious tissue damage.

Are increased frequency of macrophage-like and natural killer (NK) cells, together with high levels of NKT and CD4+CD25high T cells balancing activated CD8+ T cells, the key to control Chagas' disease morbidity?

The immunological response during early human Trypanosoma cruzi infection is not completely understood, despite its role in driving the development of distinct clinical manifestations of chronic infection. Herein we report the results of a descriptive flow cytometric immunophenotyping investigation of major and minor peripheral blood leucocyte subpopulations in T. cruzi-infected children, characterizing the early stages of the indeterminate clinical form of Chagas' disease. Our results indicated significant alterations by comparison with uninfected children, including increased values of pre-natural killer (NK)-cells (CD3- CD16+ CD56-), and higher values of proinflammatory monocytes (CD14+ CD16+ HLA-DR++). The higher values of activated B lymphocytes (CD19+ CD23+) contrasted with impaired T cell activation, indicated by lower values of CD4+ CD38+ and CD4+ HLA-DR+ lymphocytes, a lower frequency of CD8+ CD38+ and CD8+ HLA-DR+ cells; a decreased frequency of CD4+ CD25HIGH regulatory T cells was also observed. These findings reinforce the hypothesis that simultaneous activation of innate and adaptive immunity mechanisms in addition to suppression of adaptive cellular immune response occur during early events of Chagas' disease. Comparative cross-sectional analysis of these immunophenotypes with those exhibited by patients with late chronic indeterminate and cardiac forms of disease suggested that a shift toward high values of macrophage-like cells extended to basal levels of proinflammatory monocytes as well as high values of mature NK cells, NKT and regulatory T cells, may account for limited tissue damage during chronic infection favouring the establishment/maintenance of a lifelong indeterminate clinical form of the disease. On the other hand, development of an adaptive cell-mediated inflammatory immunoprofile characterized by high levels of activated CD8+ cells and basal levels of mature NK cells, NKT and CD4+ CD25HIGH cells might lead to late chronic pathologies associated with chagasic heart disease.

In vitro stimulation of human tonsillar subepithelial B cells: requirement for interaction with activated T cells.

Human tonsillar subepithelial B cells, which are a marginal zone-equivalent B cell subset, respond readily to T-independent type 2 antigens, but not to polyclonal B cell activators in vitro. In this study, subepithelial (SE) B cells were induced to proliferate and mature into plasma cells when co-cultured with activated T cells. The response of SE B cells was not observed when co-cultures were carried out in transwell chambers or in the presence of blocking anti-LFA-1 antibodies, demonstrating the need for a close T-B cell interaction. The presence of soluble CD40 also prevented the B cell response in vitro suggesting a pivotal role of CD40-CD40 ligand interactions. The data are discussed in terms of the T cell dependence of marginal zone (MZ) B cell response and the possible existence of various MZ B cell subsets.

Apoptosis or plasma cell differentiation of CD38-positive B-chronic lymphocytic leukemia cells induced by cross-linking of surface IgM or IgD.

Previously, we demonstrated that B-chronic lymphocytic leukemia (B-CLL) cells could be divided into 2 groups depending on the expression of CD38 by the malignant cells. The 2 groups differed in their signal-transducing capacities initiated by cross-linking of surface IgM; only in CD38-positive cells was an efficient signal delivered, invariably resulting in cell apoptosis. In this study, we investigated the effect of surface IgD cross-linking in 10 patients with CD38-positive B-CLL. Exposure of the malignant cells to goat antihuman delta-chain antibodies (Gadelta-ab) caused [Ca(++)]i mobilization and tyrosine kinase phosphorylation in a manner not different from that observed after goat antihuman mu-chain antibody (Gamu-ab) treatment in vitro. However, Gadelta-ab-treated cells failed to undergo apoptosis and instead displayed prolonged survival in culture and differentiated into plasma cells when rIL2 was concomitantly present. Cross-linking of surface IgD failed to induce proliferation of the malignant cells in vitro. Moreover, treatment with Gadelta-ab did not prevent apoptosis of B-CLL cells induced by Gamu-ab. Collectively, these experiments demonstrated that IgM and IgD expressed by the same cell may deliver opposite signals under particular circumstances and provide some clues for the understanding of the pathophysiology of B-CLL. (Blood. 2000;95:1199-1206)

Subepithelial B cells in the human palatine tonsil. II. Functional characterization.

This study investigates the main functional features of subepithelial (SE) B cells and compares them with those of purified germinal center (GC) and follicular mantle (FM) B cells isolated from the same tonsils. Unlike FM B cells, SE B cells failed to produce polyspecific antibodies in vitro; unlike GC B cells, SE B cells expressed high levels of Bcl-2 and failed to undergo spontaneous apoptosis in vitro. The most striking function of SE B cells was their ability to produce IgM antibodies to T cell-independent type-2 (TI-2) (but not to TI-1) antigens (Ag). These antibodies could not be detected when both FM and GC B cells were stimulated with TI-2 Ag in vitro. Moreover, B cells isolated from peripheral blood were unable to mount a response to TI-2 Ag. The latter finding is consistent with the observation that B cells with the phenotypic features of SE B cells were virtually absent in the peripheral blood and emphasizes the notion that SE B cells belong to a subset of non-recirculating B cells. SE B cells were by far superior to FM B cells in mixed lymphocyte reaction (MLR) stimulation of allogeneic T cells in vitro, although they were not as efficient as dendritic cells (DC). In order to stimulate T cells efficiently, SE B cells had to be exposed to anti-mu antibody, a treatment which induced expression of activation markers such as CD80, CD86, CD69 and CD39, usually absent in resting SE B cells. CD80 and CD86 molecules expressed by SE B cells participated in the chain of events required to promote the proliferation of allogeneic T cells as demonstrated by inhibition tests with the appropriate mAb. The expression of CD80 and CD86 by anti-mu-treated SE B cells was not, however, the sole explanation for their good antigen presenting capacities since the exposure of FM B cells to anti-mu antibody also induced expression of these surface structures. Nevertheless, these cells failed to become good MLR stimulators. Collectively, the above data contribute further to the characterization of a distinct subset of tonsillar B cells which resemble, both phenotypically and functionally, the B cells of the splenic marginal zone.

CD38 expression distinguishes two groups of B-cell chronic lymphocytic leukemias with different responses to anti-IgM antibodies and propensity to apoptosis.

The expression of CD38 by B cells chronic lymphocytic leukemia (B-CLL) was studied in 20 untreated patients. The cells expressed abundant CD38 (relative fluorescence intensity range, 6 to 15) in 6 cases (group I patients), whereas CD38 expression was low to absent (relative fluorescence intensity range, 0 to 3) in the remaining cases (group II patients). Exposure of the cells from group I patients to goat antihuman mu chain antibodies (Ga mu-ab) resulted in the elevation of intracellular free Ca2+ concentration([Ca2+]i) followed by apoptosis. In contrast, exposure of group II cells to Ga mu-ab was not followed by increased levels of [Ca2+]i, programmed cell death or cell proliferation. No differences in the expression of surface IgM were noted in the two groups of B-CLL cells. Normal peripheral blood B cells, which expressed low to absent CD38, were capable of mobilizing [Ca2+]i and of proliferating after exposure to Ga mu-ab. The collected data suggest that, although group I B-CLL cells were able to transduce the signals delivered by IgM crosslinking, this pathway was severely impaired in group II B-CLL cells. However, unlike that observed in normal circulating B cells, stimulation of group I cells with Ga mu-ab resulted in apoptosis rather than proliferation. CD38 did not appear to be directly involved in [Ca2+]i mobilization induced by Ga mu-ab in group I B-CLL cells because their exposure to anti-CD38 monoclonal antibodies failed to cause [Ca2+]i mobilization or to block the [Ca2+]i response induced by Ga mu-ab. These data indicate that CD38 expression identified a particular subset of B-CLL cells with defined functional properties, including the propensity to undergo apoptosis.

Expression of CD5 and CD38 by human CD5- B cells: requirement for special stimuli.

In this study the mode of expression of CD5 by human tonsillar CD5- B cells after stimulation with different agents was investigated. Resting B cells were separated into CD5+ and CD5- cells and the two cell fractions exposed to phorbol 12-myristate 13-acetate (PMA). CD5- B cells expressed CD5 and maximum CD5 expression was achieved after approximately 60 h of culture. Based upon the proportions of cells that express CD5 as well as those of the cells surviving in culture, it was calculated that 15-25% of the total CD5- B cells were induced to express CD5. Unlike CD5- B cells, CD5+ B cells proliferated vigorously in response to PMA as assessed by [3H]thymidine incorporation and cell cycle analysis in vitro. However, the expression of CD5 by CD5- B cells was not related to the selective expansion of some CD5+ B cells left over as contaminant cells since this occurred in the absence of cell proliferation. Upon exposure to PMA, CD5- B cells remained in the G0-G1 phases of the cell cycle and did not express the Ki67 antigen or incorporate [3H]thymidine. Furthermore, mitomycin C treatment of the CD5- B cells did not prevent CD5 expression. Phenotypic studies disclosed that CD5+ B cells but not CD5- B cells expressed CD39. This finding offered the opportunity to carry out an additional control experiment. Separation of the two populations according to the expression of CD39 confirmed the finding obtained by fractionating the cells into CD5+ and CD5- B cells. The cells induced to express CD5 also expressed CD38 that was not detected on resting CD5- B cells. In this respect, the CD5- B cells that converted into CD5+ cells (inducible CD5+ B cells) resembled the cells from the CD5+ B cell fractions that up-regulated CD5 and also expressed CD38 upon exposure to PMA alone. Another example of coordinate expression of these two antigens was the finding that exposure to PMA in the presence of recombinant interleukin-4 (rIL-4) resulted in inhibition of the expression of CD5 and CD38. Although virtually all of the tonsillar CD5- B cells expressed the CD69 activation marker, no cells other than those co-expressing CD5 and CD38 were induced to express CD5 by PMA alone. Resting CD5- B cells failed to express CD5 and/or CD38 when cultured with PMA in the presence of EL4 T cells and IL-4-free T cell supernatants.(ABSTRACT TRUNCATED AT 400 WORDS)

Coexpression of Fc gamma receptor IIIA and interleukin-2 receptor beta chain by a subset of human CD3+/CD8+/CD11b+ lymphocytes.

In this study we identify and characterize a subset of human peripheral blood T cells, present in all individuals, that has features previously described for T cells either separately or in special circumstances. These cells are found in purified suspensions of resting peripheral blood lymphocytes within the CD8+ T lymphocytes, express alpha beta T cell receptor (TCR), and can be identified and isolated because of high-density expression of surface CD11b (TCR alpha beta +/CD3+/CD8+/CD11b+ cells). They coexpress constitutively the IL-2 receptor beta chain, Fc gamma RIIIA, and CD56. Although they do not mediate spontaneous cytotoxicity, CD3+/CD8+/CD11b+ cells have cytotoxic potential, demonstrated in redirected cytotoxicity assays with P815 target cells in the presence of anti-Fc gamma RIII (CD16) or anti-CD3 monoclonal antibodies. Stimulation of CD3+/CD8+/CD11b+ cells with rIL-2 induces proliferation, cytotoxicity against NK-sensitive and NK-resistant target cells, and expression of surface activation antigens, including IL-2 receptor alpha chain (CD25). CD3+/CD8+/CD16+/CD56+ cell clones with cytotoxic functions including those mediated by engagement of surface CD16 were obtained by limiting-dilution cloning of purified CD3+/CD8+/CD11b+ cells in the presence of rIL-2 and autologous feeder cells. Our data support the hypothesis that the CD3+/CD8+/CD11b+/CD16+ cells represent a discrete peripheral blood lymphocyte subset that could be the physiological counterpart of that expanded in several pathological conditions and in large granular lymphocyte lymphocytosis.

Divergent effect of the anaerobic bacteria by-product butyric acid on the immune response: suppression of T-lymphocyte proliferation and stimulation of interleukin-1 beta production.

The effects of short-chain fatty acids (SCFA) produced by anaerobic bacteria, namely propionic, butyric and iso-butyric on T-cell proliferation was investigated. A dose-dependent inhibition of both phytohemagglutinin-induced blastogenesis and mixed lymphocyte culture was observed in the millimolar range of SCFA concentrations. The tested SCFA displayed different levels of suppression. The degree of activity was in the following order: butyrate greater than propionate greater than isobutyrate. T-cell inhibition was partially reversed, at least for propionic and isobutyric acids, by increasing the concentration of macrophages in the assay system. Furthermore, butyric acid displayed an interesting biphasic stimulation of monocyte interleukin-1 beta production, a cytokine with a powerful bone-resorbing activity. Since millimolar concentrations of SCFA are present in gingival fluid from periodontal pockets, the observed results support the role of these by-products of anaerobic metabolism in the pathogenesis of periodontal diseases.

[Inhibition of the immune response due to the volatile fatty acids produced by anaerobic bacteria in the periodontal pocket]. / Inibizione della risposta immunitaria da parte degli acidi grassi volatili prodotti dai batteri anaerobi della tasca parodontale.

Short-chain fatty acids produced by anaerobic bacteria of the periodontal pockets inhibit lymphocytes activation. The highest degrees of immunosuppressive activity is produced by butyric acid.