Physiological stress in eastern black rhinoceros (Diceros bicornis michaeli) as influenced by their density, climatological variables and sexes

It is important to understand the physiological stressors in animals especially for threatened species or intensively managed to improve their conservation and optimise their reproduction. We sought to understand changes in stress hormones (faecal glucocorticoid metabolites) in black rhinoceros (Diceros bicornis michaeli) in relation to population density and sex (intrinsic factors) and plant minerals, rainfall and land surface temperature (extrinsic factors). We used non-invasive faecal sampling techniques on animals of known sex, age and dominance in seven populations of contrasting population densities over 1 year. We measured variability in faecal corticosterone metabolites through radioimmunoassay and related them to population density, sex and faecal calcium, phosphorus, copper, zinc and potassium as characteristic of plant minerals, rainfall and temperature. We used linear mixed models (LMM) to analyse the data. We did not detect a significant relationship between physiological stress parameters and population density. However, we have indications that stress levels increased as rainfall and temperature increased and were correlated negatively and positively with concentration of faecal phosphorus and copper respectively; we found higher stress levels in females than in males. These results suggest that both intrinsic and extrinsic factors explain the variation in physiological stress observed in black rhinoceros

Plasma angiopoietin-2 is associated with age-related deficits in cognitive sub-scales in Ugandan children following severe malaria

Background: Elevated angiopoietin-2 (Angpt-2) concentrations are associated with worse overall neurocognitive function in severe malaria survivors, but the specific domains affected have not been elucidated. Methods: Ugandan children with severe malaria underwent neurocognitive evaluation a week after hospital discharge and at 6, 12 and 24 months follow-up. The relationship between Angpt-2 concentrations and age-adjusted, cognitive sub-scale z-scores over time were evaluated using linear mixed effects models, adjusting for disease severity (coma, acute kidney injury, number of seizures in hospital) and sociodemographic factors (age, gender, height-for-age z-score, socio-economic status, enrichment in the home environment, parental education, and any preschool education of the child). The Mullen Scales of Early Learning was used in children < 5 years and the Kaufman Assessment Battery for Children 2nd edition was used in children ≥ 5 years of age. Angpt-2 levels were measured on admission plasma samples by enzyme-linked immunosorbent assay. Adjustment for multiple comparisons was conducted using the Benjamini-Hochberg Procedure of False Discovery Rate. Results: Increased admission Angpt-2 concentration was associated with worse outcomes in all domains (fine and gross motor, visual reception, receptive and expressive language) in children < 5 years of age at the time of severe malaria episode, and worse simultaneous processing and learning in children < 5 years of age at the time of severe malaria who were tested when ≥ 5 years of age. No association was seen between Angpt-2 levels and cognitive outcomes in children ≥ 5 years at the time of severe malaria episode, but numbers of children and testing time points were lower for children ≥ 5 years at the time of severe malaria episode. Conclusion: Elevated Angpt-2 concentration in children with severe malaria is associated with worse outcomes in multiple neurocognitive domains. The relationship between Angpt-2 and worse cognition is evident in children < 5 years of age at the time of severe malaria presentation and in selected domains in older years

Endothelial Activation, Acute Kidney Injury, and Cognitive Impairment in Pediatric Severe Malaria

Objectives: Evaluate the relationship between endothelial activation, malaria complications, and long-term cognitive outcomes in severe malaria survivors. Design: Prospectively cohort study of children with cerebral malaria, severe malarial anemia, or community children. Setting: Mulago National Referral Hospital in Kampala, Uganda. Subjects: Children 18 months to 12 years old with severe malaria (cerebral malaria, n = 253 or severe malarial anemia, n = 211) or community children (n = 206) were followed for 24 months. Interventions: None. Measurements and main results: Children underwent neurocognitive evaluation at enrollment (community children) or a week following hospital discharge (severe malaria) and 6, 12, and 24 months follow-up. Endothelial activation was assessed at admission on plasma samples (von Willebrand factor, angiopoietin-1 and angiopoietin-2, soluble intercellular adhesion molecule-1, soluble vascular cell adhesion molecule-1, soluble E-Selectin, and P-Selectin). False discovery rate was used to adjust for multiple comparisons. Severe malaria was associated with widespread endothelial activation compared with community children (p < 0.0001 for all markers). Acute kidney injury was independently associated with changes in von Willebrand factor, soluble intercellular adhesion molecule-1, soluble E-Selectin, P-Selectin, and angiopoietin-2 (p < 0.0001 for all). A log10 increase in angiopoietin-2 was associated with lower cognitive z scores across age groups (children < 5, β -0.42, 95% CI, -0.69 to -0.15, p = 0.002; children ≥ 5, β -0.39, 95% CI, -0.67 to -0.11, p = 0.007) independent of disease severity (coma, number of seizures, acute kidney injury) and sociodemographic factors. Angiopoietin-2 was associated with hemolysis (lactate dehydrogenase, total bilirubin) and inflammation (tumor necrosis factor-α, interleukin-10). In children with cerebral malaria who had a lumbar puncture performed, angiopoietin-2 was associated with blood-brain barrier dysfunction, and markers of neuroinflammation and injury in the cerebrospinal fluid (tumor necrosis factor-α, kynurenic acid, tau). Conclusions: These data support angiopoietin-2 as a measure of disease severity and a risk factor for long-term cognitive injury in children with severe malaria

Tracking the Feeding Patterns of Tsetse Flies (Glossina Genus) by Analysis of Bloodmeals Using Mitochondrial Cytochromes Genes

Tsetse flies are notoriously difficult to observe in nature, particularly when populations densities are low. It is therefore difficult to observe them on their hosts in nature; hence their vertebrate species can very often only be determined indirectly by analysis of their gut contents. This knowledge is a critical component of the information on which control tactics can be developed. The objective of this study was to determine the sources of tsetse bloodmeals, hence investigate their feeding preferences. We used mitochondrial cytochrome c oxidase 1 (COI) and cytochrome b (cytb) gene sequences for identification of tsetse fly blood meals, in order to provide a foundation for rational decisions to guide control of trypanosomiasis, and their vectors. Glossina swynnertoni were sampled from Serengeti (Tanzania) and G. pallidipes from Kenya (Nguruman and Busia), and Uganda. Sequences were used to query public databases, and the percentage identities obtained used to identify hosts. An initial assay showed that the feeds were from single sources. Hosts identified from blood fed flies collected in Serengeti ecosystem, included buffaloes (25/40), giraffes (8/40), warthogs (3/40), elephants (3/40) and one spotted hyena. In Nguruman, where G. pallidipes flies were analyzed, the feeds were from elephants (6/13) and warthogs (5/13), while buffaloes and baboons accounted for one bloodmeal each. Only cattle blood was detected in flies caught in Busia and Uganda. Out of four flies tested in Mbita Point, Suba District in western Kenya, one had fed on cattle, the other three on the Nile monitor lizard. These results demonstrate that cattle will form an integral part of a control strategy for trypanosomiasis in Busia and Uganda, while different approaches are required for Serengeti and Nguruman ecosystems, where wildlife abound and are the major component of the tsetse fly food source

Sex and Urbanicity Contribute to Variation in Lymphocyte Distribution across Ugandan Populations.

Management of patient care and interpretation of research data require evaluation of laboratory results in the context of reference data from populations with known health status to adequately diagnose disease or make a physiological assessment. Few studies have addressed the diversity of lymphocyte subsets in rural and urban Ugandan populations. Here, 663 healthy blood bank donors from semi-urban centers of Kampala consented to participate in a study to define lymphocyte reference ranges. Whole blood immunophenotyping was performed to determine the frequency and absolute counts of T, B, and NK cells using clinical flow cytometry. Results from blood bank donors were compared to a rural cohort from the district of Kayunga and more urban clinical trial participants from the capital city, Kampala. Relationships between hematological and lymphocyte parameters were also explored. In the semi-urban blood donors, females were significantly different from males in all parameters except the frequency of CD8 T and B cells. Females had higher absolute counts of CD4 T, CD8 T and B cells, whereas males had higher NK cell counts. NK cell frequency and counts were significantly higher in semi-urban blood donors, regardless of sex, compared to more urban study participants. CD8 T cell frequency and counts were significantly higher in the blood donors compared to the rural participants, irrespective of sex. Interestingly, basophil counts were positively associated with overall T cell counts. These findings suggest that both sex and level of cohort urbanicity may influence lymphocyte subset distributions in Ugandans

Quality Monitoring of HIV-1-Infected and Uninfected Peripheral Blood Mononuclear Cell Samples in a Resource-Limited Setting▿

Human immunodeficiency virus type 1 (HIV-1) vaccine and natural history studies are critically dependent on the ability to isolate, cryopreserve, and thaw peripheral blood mononuclear cell (PBMC) samples with a high level of quality and reproducibility. Here we characterize the yield, viability, phenotype, and function of PBMC from HIV-1-infected and uninfected Ugandans and describe measures to ascertain reproducibility and sample quality at the sites that perform cryopreservation. We have developed a comprehensive internal quality control program to monitor processing, including components of method validation. Quality indicators for real-time performance assessment included the time from venipuncture to cryopreservation, time for PBMC processing, yield of PBMC from whole blood, and viability of the PBMC before cryopreservation. Immune phenotype analysis indicated lowered B-cell frequencies following processing and cryopreservation for both HIV-1-infected and uninfected subjects (P < 0.007), but all other major lymphocyte subsets were unchanged. Long-term cryopreservation did not impact function, as unstimulated specimens exhibited low background and all specimens responded to staphylococcal enterotoxin B (SEB) by gamma interferon and interleukin-2 production, as measured by intracellular cytokine staining. Samples stored for more than 3 years did not decay with regard to yield or viability, regardless of HIV-1 infection status. These results demonstrate that it is possible to achieve the high level of quality necessary for vaccine trials and natural history studies in a resource-limited setting and provide strategies for laboratories to monitor PBMC processing performance

A High Throughput Whole Blood Assay for Analysis of Multiple Antigen-Specific T Cell Responses in Human Mycobacterium tuberculosis Infection

Antigen-specific CD4 and CD8 T cells are important components of the immune response to Mycobacterium tuberculosis, yet little information is currently known regarding how the breadth, specificity, phenotype, and function of M. tuberculosis-specific T cells correlate with M. tuberculosis infection outcome in humans. To facilitate evaluation of human M. tuberculosis-specific T cell responses targeting multiple different Ags, we sought to develop a high throughput and reproducible T cell response spectrum assay requiring low blood sample volumes. We describe here the optimization and standardization of a microtiter plate-based, diluted whole blood stimulation assay utilizing overlapping peptide pools corresponding to a functionally diverse panel of 60 M. tuberculosis Ags. Using IFN-γ production as a readout of Ag specificity, the assay can be conducted using 50 μl of blood per test condition and can be expanded to accommodate additional Ags. We evaluated the intra- and interassay variability, and implemented testing of the assay in diverse cohorts of M. tuberculosis-unexposed healthy adults, foreign-born adults with latent M. tuberculosis infection residing in the United States, and tuberculosis household contacts with latent M. tuberculosis infection in a tuberculosis-endemic setting in Kenya. The M. tuberculosis-specific T cell response spectrum assay further enhances the immunological toolkit available for evaluating M. tuberculosis-specific T cell responses across different states of M. tuberculosis infection, and can be readily implemented in resource-limited settings. Moreover, application of the assay to longitudinal cohorts will facilitate evaluation of treatment- or vaccine-induced changes in the breadth and specificity of Ag-specific T cell responses, as well as identification of M. tuberculosis-specific T cell responses associated with M. tuberculosis infection outcomes