Parasite Community Analysis of The Gray Snapper Lutjanus Griseus (Perciformes,Lutjanidae) in a Tropical Region of The Southern Gulf of Mexico.

The gray snapper Lutjanus griseus is a commercially important fish species along its distribution range in the western Atlantic Ocean. However, despite its importance, there is still little knowledge about its parasitic fauna for the Mexican coasts of the Gulf of Mexico. The aims of this research were to generate a list of the parasitic fauna present in juvenile gray snapper L. griseus from a coastal lagoon located in southeastern Mexico, to evaluate the infection levels of parasites and to determine the relationship between the abundance of parasites and the fish size and condition factor. Samples of L. griseus (12 - 29.2 mm) were obtained in two periods of the year (dry and rainy seasons) to examine the intra-annual variability of its parasitic fauna. A total of 17 parasite species were recorded belonging to six taxonomic groups (Myxozoa, Monogenea, Digenea, Cestoda, Nematoda and Acanthocephala). The highest levels of infection (abundance, prevalence and intensity of infection) were found for the monogeneans Euryhaliotrema griseus and Euryhaliotrema fastigatum. There were no significant correlations between the total abundance of parasites and the fish condition and size (total length) in not any of the two seasons studied, suggesting that the body size and the biological condition index of the host did not directly influence the abundance of parasites in early life stages of L. griseus. Moreover, the species of parasites found that could be zoonotic for humans through the consumption of raw or inadequately cooked fish were the nematodes Contracaecum sp. type 1, Contracaecum sp. type 2, Cucullanus pargi and Pseudoterranova sp. The presence of the monogeneans E. griseus and E. fastigatum was also highlighted because these ectoparasite species are known to cause harm to fish under culture systems. All the parasite species found in this study, except nematodes, were new records of geographic distribution.

Economic evaluation of the environmental impact of a dairy cattle intensive production cluster under arid lands conditions.

At a global level, dairy cow production systems (DCPS) are important sources of nourishment and profits, but they generate environmental impacts such as overexploitation of different resources including water, lands and fossil energy. Quantification of water and carbon footprint to define mitigation strategies and a more rational use of natural resources, is a reiterated claim. The aim of this study was to perform an economic evaluation of the environmental impact of the DCPS from the Comarca Lagunera, Mexico (24°N, 102°W, 220 mm, hot-semiarid climate) We contrasted the economic value (EV) generated by the DCPS with respect to the economic costs (EC) due to the greenhouse gas emissions (GHGE) and the water footprint (WFP) of this DCPS. While quantifications of GHGE considered those proposed by the Intergovernmental Panel on Climate Change, the WFP involved the use of blue, gray and green water by the DCPS and related activities. Quantification of the EC of WFP considered an international average price of water. In the year 2017, the Comarca Lagunera registered a dairy cow inventory of 493 144 heads, with 227 142 lactating cows, which produced 2386 million liters of milk per year with an annual average EV of €525.3 million. The EC (€, millions) generated by the GHGE and WFP were €311.8 and €11 980.7, respectively, with a total EC of € 12 292.5 million. When the EV of milk production and the total environmental EC are compared, the contrast demonstrates not only the noteworthy environmental impact but also the significant and senseless biological and EC. In addition, having a large dairy cow concentration creates pollution concerns and the DCPS transfers both nutrients and water resources from an ecologically vulnerable arid region. Therefore, some mitigation strategies such as, better cow genotype, feed and manure management combined with the production of forages and grains in a different geographical region are suggested to promote an optimum use of water in order to uphold the social, economic and biologic sustainability of the Comarca Lagunera, Mexico.

Link Between the Adult and the Metacercaria of Clinostomum heluans Braun, 1899 (Trematoda: Clinostomidae) Through DNA Sequences, and its Phylogenetic Position Within the Genus Clinostomum Leidy, 1856.

The phylogenetic position of Clinostomum heluans Braun, 1899 within the genus Clinostomum Leidy, 1856 is reported in this study based on sequences of the barcoding region of the mitochondrial cytochrome c oxidase subunit 1 gene ( COX1). Additionally, molecular data are used to link the adult and the metacercariae of the species. The metacercariae of C. heluans were found encysted infecting the cichlid fish Australoheros sp. in Minas Gerais, Brazil, whereas the adults were obtained from the mouth cavity of the Great White Egret, Ardea alba, in Campeche, Mexico. The COX1 sequences obtained for the Mexican clinostomes and the Brazilian metacercaria were almost identical (0.2% molecular divergence), indicating conspecificity. Similar molecular divergence (0.2-0.4%) was found between sequences of C. heluans reported here and Clinostomum sp. 6 previously obtained from a metacercaria recovered from the cichlid Cichlasoma boliviense in Santa Cruz, Bolivia. Both maximum likelihood and Bayesian inference analyses unequivocally showed the conspecificity between C. heluans and Clinostomum sp. 6, which form a monophyletic clade with high nodal support and very low genetic divergence. Moreover, tree topology reveals that C. heluans occupies a basal position with respect to New World species of Clinostomum, although a denser taxon sampling of species within the genus is further required. The metacercaria of C. heluans seems to be specific to cichlid fish because both samples from South America were recovered from species of this fish family, although not closely related.

Randomized, double-blind, placebo-controlled study on the immunogenicity of the leishmanin skin test.

A positive reaction to the leishmanin skin test (LST) indicates previous contact with Leishmania antigens and is a useful criterion for the diagnosis of cutaneous leishmaniasis. In leishmaniasis vaccine trials, selection of volunteers has always been based on skin testing. During 1999 we performed a randomized controlled study in order to evaluate the immunogenicity of the LST. Fifty-nine (29 male and 30 female) healthy volunteer undergraduate students from the Medical School of Volta Redonda, Rio de Janeiro State, Brazil, with no evidence of previous infection with Leishmania, were randomly assigned into 2 groups: 29 subjects received LST and 30 received a placebo (merthiolate-phosphate-buffered saline). All volunteers received LST 41 d after the first injection of LST or placebo. Blood samples were taken immediately before the applications of LST or placebo for the assessment of Leishmania antigen-induced proliferation and cytokine production in peripheral blood mononuclear cell cultures. A significant increase in proliferative responses to L. braziliensis (P < 0.005) and L. amazonensis (P = 0.01) antigens as well as in L. braziliensis antigen-induced interferon-gamma production (P < 0.01) followed the application of LST but not the administration of the placebo. A single LST application is therefore able to induce Leishmania-specific cell-mediated immune responses. This observation should be considered in human trials of candidate vaccines against leishmaniasis.

A randomized double-blind placebo-controlled trial to evaluate the immunogenicity of a candidate vaccine against American tegumentary leishmaniasis.

This study was aimed at evaluating the immunogenicity of a vaccine composed of killed Leishmania amazonensis promastigotes using several different protocols in a randomized, double-blind and controlled trial design in order to select one of them for further efficacy trials. One hundred and fourteen leishmanin skin test (LST)-negative healthy volunteers were allocated into eight groups that received either two or three deep intramuscular injections of vaccine at doses of 180, 360 and 540 microg or similar injections of placebo. Cell-mediated immune responses were evaluated before and after vaccination by means of LST as well as proliferative responses and cytokine production in Leishmania antigen-stimulated peripheral blood mononuclear cell cultures. The majority of the subjects who actually received vaccine converted to positive LST (89.5%). On the other hand, none of the subjects who received placebo converted to positive LST. Proliferative responses and production of interferon-gamma and interleukin-2 were significantly higher after vaccination than before vaccination in all groups, including those that received placebo. The dose of 360 microg provided the highest LST conversion rate (100%), as well as the greatest increase in interferon-gamma and interleukin-2 production after vaccination.

Lack of association between intussusception and oral polio vaccine in Cuban children.

Two exploratory investigations found an increased risk of intussusception after oral polio vaccine (OPV). A large, national, population-based study was undertaken in Cuba to investigate a possible association. Three hundred and thirty-five cases of intussusception in children under 2 years of age occurring in 1995-2000 were identified and their OPV records retrieved. The relative incidence (RI) of intussusception in defined periods up to 42 days after OPV in children under 1 year was estimated using the self-controlled case series method, controlling for age and season. The RI was not significantly raised in any of the time intervals examined within the 0-42 day period after OPV. For the period 0-42 days as a whole the RI was 1.11, 95% CI 0.74-1.67. This study does not support the hypothesis that OPV causes intussusception.

Characterisation of lymphocyte response and cytokine patterns in patients with dengue fever.

It is believed that the pathogenesis of dengue is generated by a deregulation of the immunological response. Dengue virus-infected monocytes/macrophages are likely to secrete monokines, which play a role in clinical features observed in patients with dengue haemorrhagic fever or dengue shock syndrome. This is a report on a study on 45 individuals presenting clinical and laboratory characteristics of dengue virus infection. During the acute phase of infection, immunophenotyping of peripheral mononuclear leukocytes was carried out in 19 patients and demonstrated a reduced frequency of CD2+ lymphocytes and their CD4+ and CD8+ subsets. Normal ratios were recovered during convalescence. Also, during the acute phase, mononuclear cells proliferated poorly in response to mitogens and dengue antigens as detected by incorporation of radiolabeled thymidine. During convalescence the lymphoproliferative response was re-established. In addition, the presence of circulating cytokines was investigated in the plasma of the same 45 patients. Concentrations of tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-10 (IL-10) and soluble tumor necrosis factor receptor (sTNF-Rp75) were found to be significantly elevated in patients when compared to normal controls. The increase in TNF-alpha was correlated with haemorrhagic manifestations and the increase in IL-10 with platelet decay. The data demonstrate that during the acute phase of dengue infection subsets of T lymphocytes are depressed in terms of both rate and function and provide evidence that circulating pro-inflammatory cytokines, such as TNF-alpha, are important in the pathogenesis and severity of dengue. IL-10 may be downregulating lymphocyte and platelet function.

Comparison of dengue infection in human mononuclear leukocytes with mosquito C6/36 and mammalian Vero cells using flow cytometry to detect virus antigen.

Fluorescent activated cell sorter (FACS) analysis is useful for the detection of cellular surface antigens and intracellular proteins. We used this methodology in order to detect and quantify dengue antigens in highly susceptible cells such as clone C6/36 (Aedes albopictus) and Vero cells (green monkey kidney). Additionally, we analyzed the infection in vitro of human peripheral blood mononuclear leukocytes (PBML). FACS analysis turned out to be a reliable technique to quantify virus growth in traditional cell cultures of C6/36 as well as Vero cells. High rates of infection were achieved with a good statistical correlation between the virus amount used in infection and the percentage of dengue antigen containing cells detected in infected cultures. We also showed that human monocytes (CD14+) are preferred target cells for in vitro dengue infection among PBML. Monocytes were much less susceptible to virus infection than cell lines but they displayed dengue antigens detected by FACS five days after infection. In contrast, lymphocytes showed no differences in their profile for dengue specific immunofluorescence. Without an animal model to reproduce dengue disease, alternative assays have been sought to correlate viral virulence with clinical manifestations and disease severity. Study of in vitro interaction of virus and host cells may highlight this relationship.

Flow cytometry in the study of cell death.

In this report we present a concise review concerning the use of flow cytometric methods to characterize and differentiate between two different mechanisms of cell death, apoptosis and necrosis. The applications of these techniques to clinical and basic research are also considered. The following cell features are useful to characterize the mode of cell death: (1) activation of an endonuclease in apoptotic cells results in extraction of the low molecular weight DNA following cell permeabilization, which, in turn, leads to their decreased stainability with DNA-specific fluorochromes. Measurements of DNA content make it possible to identify apoptotic cells and to recognize the cell cycle phase specificity of apoptotic process; (2) plasma membrane integrity, which is lost in necrotic but not in apoptotic cells; (3) the decrease in forward light scatter, paralleled either by no change or an increase in side scatter, represent early changes during apoptosis. The data presented indicate that flow cytometry can be applied to basic research of the molecular and biochemical mechanisms of apoptosis, as well as in the clinical situations, where the ability to monitor early signs of apoptosis in some systems may be predictive for the outcome of some treatment protocols.

Detection of intracytoplasmic cytokines by flow cytometry.

Flow cytometry has been used as a powerful technique for studying cell surface antigen expression as well as intracellular molecules. Its capability of analyzing multiple parameters simultaneously on a single cell has allowed identification and studies of functional cell subsets within heterogeneous populations. In this respect, several techniques have been developed during the past few years to study cytokine-producing cells by flow cytometry in humans and several animal models.

Inducible nitric oxide synthase (iNOS) expression in liver and splenic T lymphocyte rise are associated with liver histological damage during experimental hepatitis A virus (HAV) infection in Callithrix jacchus.

Callithrix jacchus is considered a reliable animal model for hepatitis A virus (HAV) infection. All three HAV orally inoculated marmosets developed hepatitis - the infection was monitored by continuous virus shedding, high levels of serum enzyme alanine aminotransferase, specific antibody and seroconversion 3-6 weeks after HAV inoculation. HAV antigen was detected in liver by immunofluorescence 4 days post inoculation (PI) and onwards. To gain insight into the biological role of inducible nitric oxide synthase (iNOS) during immune-related acute liver injury the enzyme was searched in frozen biopsies: immunofluorescent labeling was found in the cytoplasm of liver cells mainly Kupffer's cells and spleen macrophages (CD68+) starting 11 days PI with maximum intensity on the fifth to sixth week PI. Necroinflammatory liver lesions characteristic of viral hepatitis were also observed at 10 days PI with maximum severity at 4 to 6 weeks PI. Furthermore, T lymphocytes (CD2+) were raised at this time point. No difference was evident in the frequency of B lymphocytes (CD20+). Therefore, iNOS expression preceded necroinflammatory liver lesion and maximal immunofluorescence reaction was coincident with tissue injury, supporting the hypothesis that NO contributes to hepatic cytotoxic mechanism but also to virus clearance. The concomitant rise in T-lymphocyte population may suggest a role for these cells in this and/or other independent HAV-induced pathological changes.

Detection of early apoptosis and cell death in T CD4+ and CD8+ cells from lesions of patients with localized cutaneous leishmaniasis.

Human localized cutaneous leishmaniasis (LCL), induced by Leishmania braziliensis, ranges from a clinically mild, self-healing disease with localized cutaneous lesions to severe forms which can present secondary metastatic lesions. The T cell-mediated immune response is extremely important to define the outcome of the disease; however, the underlying mechanisms involved are not fully understood. A flow cytometric analysis of incorporation of 7-amino actinomycin D and CD4+ or CD8+ T cell surface phenotyping was used to determine whether different frequencies of early apoptosis or accidental cell death occur at different stages of LCL lesions. When all cells obtained from a biopsy sample were analyzed, larger numbers of early apoptotic and dead cells were observed in lesions from patients with active disease (mean = 39.5 +/- 2.7%) as compared with lesions undergoing spontaneous healing (mean = 17.8 +/- 2.2%). Cells displaying normal viability patterns obtained from active LCL lesions showed higher numbers of early apoptotic events among CD8+ than among CD4+ T cells (mean = 28.5 +/- 3.8 and 15.3 +/- 3.0%, respectively). The higher frequency of cell death events in CD8+ T cells from patients with LCL may be associated with an active form of the disease. In addition, low frequencies of early apoptotic events among the CD8+ T cells were observed in two patients with self-healing lesions. Although the number of patients in the latter group was small, it is possible to speculate that, during the immune response, differences in apoptotic events in CD4+ and CD8+ T cell subsets could be responsible for controlling the CD4/CD8 ratio, thus leading to healing or maintenance of disease.

Detection of early apoptosis and cell death in T CD4+ and CD8+ cells from lesions of patients with localized cutaneous leishmaniasis

Human localized cutaneous leishmaniasis (LCL), induced by Leishmania braziliensis, ranges from a clinically mild, self-healing disease with localized cutaneous lesions to severe forms which can present secondary metastatic lesions. The T cell-mediated immune response is extremely important to define the outcome of the disease; however, the underlying mechanisms involved are not fully understood. A flow cytometric analysis of incorporation of 7-amino actinomycin D and CD4+ or CD8+ T cell surface phenotyping was used to determine whether different frequencies of early apoptosis or accidental cell death occur at different stages of LCL lesions. When all cells obtained from a biopsy sample were analyzed, larger numbers of early apoptotic and dead cells were observed in lesions from patients with active disease (mean = 39.5 + or - 2.7 per cent) as compared with lesions undergoing spontaneous healing (mean = 17.8 + or - 2.2 per cent). Cells displaying normal viability patterns obtained from active LCL lesions showed higher numbers of early apoptotic events among CD8+ than among CD4+ T cells (mean = 28.5 + or - 3.8 and 15.3 + or - 3.0 per cent, respectively). The higher frequency of cell death events in CD8+ T cells from patients with LCL may be associated with an active form of the disease. In addition, low frequencies of early apoptotic events among the CD8+ T cells were observed in two patients with self-healing lesions. Although the number of patients in the latter group was small, it is possible to speculate that, during the immune response, differences in apoptotic events in CD4+ and CD8+ T cell subsets could be responsible for controlling the CD4/CD8 ratio, thus leading to healing or maintenance of disease.

Immunologic patterns associated with cure in human American cutaneous leishmaniasis.

Patients with American cutaneous leishmaniasis were studied before therapy (active lesion) and at the end of therapy (cured patients). Assays of lymphocyte proliferative responses of peripheral blood mononuclear cells induced in vitro by Leishmania braziliensis promastigote antigens (Lb) were performed. Antigen-stimulated cells were harvested for CD4 and CD8 phenotype analysis and the levels of gamma interferon (IFN-gamma) and interleukin 4 (IL-4) produced were also determined in the culture supernatants. Two different patterns of Lb-induced T cell responses were observed: a) predominance of responding CD4+ cells and mixed type 1 and type 2 cytokine production (IFN-gamma and IL-4) during the active disease, and b) similar proportions of responding CD4+ and CD8+ cells, and type 1 cytokine production (presence of IFN-gamma and very low IL-4) at the end of therapy (healed lesions). This last pattern is probably associated with a beneficial T cell response.

Immunologic patterns associated with cure in human American cutaneous leishmaniasis

Patients with American cutaneous leishmaniasis were studied before therapy (active lesion) and at the end of therapy (cured patients). Assays of lymphocyte proliferative responses of peripheral blood mononuclear cells induced in vitro by Leishmania braziliensis promastigote antigens (Lb) were performed. Antigen-stimulated cells were harvested for CD4 and CD8 phenotype analysis and the levels of gamma interferon (IFN-gamma) and interleukin 4 (IL-4) produced were also determined in the culture supernatants. Two different patterns of Lb-induced T cell responses were observed: a) predominance of responding CD4+ cells and mixed type 1 and type 2 cytokine production (IFN-gamma and IL-4) during the active disease, and b) similar proportions of responding CD4+ and CD8+ cells, and type 1 cytokine production (presence of INF-gamma and very low IL-4) at the end of therapy (healed lesions). This last pattern is probably associated with a beneficial T cell response.

An experimental model of the production of metastases in murine cutaneous leishmaniasis.

An experimental investigation into the influence of artificially induced trauma in the production of leishmanial metastatic lesions and into the possible role played by Leishmania-reactive T cell populations in the metastatic process was carried out. Trauma was induced by incising a small cut into the shaved rump of Leishmania amazonensis-infected BALB/c mice. Ten days after the trauma, mice were killed to quantify the parasite load in the traumatic lesion or in the equivalent area in nontraumatized mice, by limiting dilution analysis. Results demonstrated that metastatic lesions occurred earlier in traumatized animals and that parasites could be detected sooner in traumatic lesions than in equivalent areas in nontraumatized mice. When lymph node cells from L. amazonensis antigen-immunized BALB/c mice were adoptively transferred intravenously to L. amazonensis-infected syngeneic mice, the parasite load in the metastatic lesions was greater in the animals that received La Ag-reactive T cells than in the controls. When CD4(+)- or CD8(+)-depleted T cell populations from La Ag-immunized mice were adoptively transferred to infected traumatized or nontraumatized animals, we observed that the metastatic lesions in CD4(+)-inoculated animals had a greater number of parasites than the lesions in mice from all other groups. Thus, a new and reliable mouse model for studying the mechanisms involved in leishmanial metastasis is described.