Embryonic thermal challenge is associated with increased stressor resiliency later in life: Molecular and morphological mechanisms in the small intestine.

Developing chick embryos that are subjected to increased incubation temperature are more stressor-resilient later in life, but the underlying process is poorly understood. The potential mechanism may involve changes in small intestine function. In this study, we determined behavioral, morphological, and molecular effects of increased embryonic incubation temperatures and post-hatch heat challenge in order to understand how embryonic heat conditioning (EHC) affects gut function. At 4 days post-hatch, duodenum, jejunum, and ileum samples were collected at 0, 2, and 12 h relative to the start of heat challenge. In EHC chicks, we found that markers of heat and oxidative stress were generally lower while those of nutrient transport and antioxidants were higher. Temporally, gene expression changes in response to the heat challenge were similar in control and EHC chicks for markers of heat and oxidative stress. Crypt depth was greater in control than EHC chicks at 2 h post-challenge, and the villus height to crypt depth ratio increased from 2 to 12 h in both control and EHC chicks. Collectively, these results suggest that EHC chicks might be more energetically efficient at coping with thermal challenge, preferentially allocating nutrients to other tissues while protecting the mucosal layer from oxidative damage. These results provide targets for future studies aimed at understanding the molecular mechanisms underlying effects of embryonic heat exposure on intestinal function and stressor resiliency later in life.

Diurnal questing behavior of Amblyomma mixtum (Acari: Ixodidae).

The diurnal questing behavior of Amblyomma mixtum Koch was monitored in the laboratory while held on a 12:12 diurnal cycle, and outdoors, using actographic chambers to determine the daily incidences of questing for a host. Nymphs and adults increased their questing activity during the morning. Adult activity peaked in the morning and continued until afternoon, whereas nymphal activity peaked in the afternoon. Nymphs and adults returned to the bottom of the chambers at night. The questing pattern for nymphs held outdoors was similar to that observed indoors. Both nymphs and adults had a higher average questing height when held at 95% RH than at 56% RH. The increase in average questing height was found to be due to fewer ticks questing-and for adults, a decrease in the mean questing height-at lower RH.

Vector potential and population dynamics for Amblyomma inornatum.

We studied the natural life cycle of Amblyomma inornatum and its vector potential in South Texas. This tick is distributed throughout South Texas and most of Central America. A. inornatum represented 1.91% of the ticks collected by carbon dioxide traps during a study of free-living ticks in the Tamaulipan Biotic Province in South Texas. The life cycle of A. inornatum in South Texas showed a clear seasonal pattern consistent with one generation per year. Nymphs emerged in the spring with a peak in February through May. Adults emerged in the summer with a peak in July through September. Detection of A. inornatum larvae was negatively correlated with saturation deficit and positively correlated with rain in the previous few months. Adult activity was positively correlated with temperature and rain in the previous five weeks. Using PCR we detected the presence of species related to Candidatus Borrelia lonestari, Borrelia burgdorferi, Rickettsia species (Candidatus Rickettsia amblyommii), Ehrlichia chaffeensis, and another Ehrlichia related to Ehrlichia ewingii. Finally we sequenced the mitochondrial 16S rRNA genes and found that A. inornatum is most closely related to Amblyomma parvum. This is the first report of the life cycle, vector potential and phylogeny of A. inornatum.

Meteorological indicators for Amblyomma cajennense and population dynamics in the Tamaulipan Biotic Province in Texas.

We studied the population dynamics of free-living ticks in the Tamaulipan Biotic Province in south Texas from March, 2005 to November, 2008. We collected 70,873 ticks using carbon dioxide traps. Amblyomma cajennense represented 93.6% of the ticks identified. A. cajennense is distributed from northern Argentina to south Texas in the Tamaulipan Biotic Province. Emergence of larval A. cajennense ticks was observed two to five weeks after significant rain events (p<0.0001) and had a strong negative correlation with temperature (p<0.0001). More larvae were observed under humid conditions (p<0.05). Fewer larvae were observed during windy and warmer conditions (p<0.05). This observation indicates high sensitivity of larvae to desiccating conditions. Peaks in nymphal activity were observed after peaks of larval emergence. Activity of nymphs was negatively correlated with temperature (p<0.05). Adult activity was negatively correlated with humidity (p<0.05) and negatively correlated with total rain from three to six weeks prior to observation (p<0.05). Adult A. cajennense are particularly tolerant to drier conditions relative to other closely related ticks. Adult female activity was positively correlated with temperature (p<0.05). Peaks in rain activity and a summer behavioral diapause appear to be the dominant factors controlling emergence of larvae, and by extension, the life cycle of A. cajennense in the Tamaulipan Biotic Province.

High prevalence of Entamoeba moshkovskii in a Tanzanian HIV population.

Entamoeba moshkovskii and Entamoeba dispar are microscopically indistinguishable from the pathogenic species Entamoeba histolytica. There are limited data on the prevalence of these commensal infections from Africa. We utilized PCR and antigen detection to evaluate the carriage rate of E. moshkovskii, E. dispar, and E. histolytica infection in stool from a cohort of HIV-suspected or confirmed inpatients from Tanzania. E. histolytica was detected by ELISA in 4% (5/118) while E. moshkovskii and E. dispar were detected by PCR in 13% (18/136) and 5% (7/136) of individuals, respectively (P<0.05). Supporting their commensal nature, neither E. moshkovskii nor E. dispar infection was statistically associated with HIV status, CD4 count, or the presence of diarrhea. These data suggest E. moshkovskii is a common infection in HIV-infected individuals in northern Tanzania and supports the concept that the microscopic detection of Entamoeba should be interpreted cautiously.

Identification and gene expression analysis of a large family of transmembrane kinases related to the Gal/GalNAc lectin in Entamoeba histolytica.

We identified in the Entamoeba histolytica genome a family of over 80 putative transmembrane kinases (TMKs). The TMK extracellular domains had significant similarity to the intermediate subunit (Igl) of the parasite Gal/GalNAc lectin. The closest homolog to the E. histolytica TMK kinase domain was a cytoplasmic dual-specificity kinase, SplA, from Dictyostelium discoideum. Sequence analysis of the TMK family demonstrated similarities to both serine/threonine and tyrosine kinases. TMK genes from each of six phylogenetic groups were expressed as mRNA in trophozoites, as assessed by spotted oligoarray and real-time PCR assays, suggesting nonredundant functions of the TMK groups for sensing and responding to extracellular stimuli. Additionally, we observed changes in the expression profile of the TMKs in continuous culture. Antisera produced against the conserved kinase domain identified proteins of the expected molecular masses of the expressed TMKs. Confocal microscopy with anti-TMK kinase antibodies revealed a focal distribution of the TMKs on the cytoplasmic face of the trophozoite plasma membrane. We conclude that E. histolytica expresses members of each subgroup of TMKs. The presence of multiple receptor kinases in the plasma membrane offers for the first time a potential explanation of the ability of the parasite to respond to the changing environment of the host.

In vitro and in vivo interaction of Entamoeba histolytica Gal/GalNAc lectin with various target cells: an immunocytochemical analysis.

The Gal/GalNAc lectin of Entamoeba histolytica trophozoites plays an important role in adhesion. The distribution and final destiny of the lectin during the interaction with host cells are poorly understood. Using monoclonal and polyclonal antibodies against the lectin we studied by immunocytochemistry the in vitro and in vivo interaction of E. histolytica trophozoites with human and hamster hepatocytes. We also analyzed the presence and distribution of the lectin in a mouse model of intestinal amoebiasis. In all cases, trophozoites were highly labeled by anti-lectin antibodies. Cultured human and hamster hepatocytes in contact with, or localized at the vicinity of parasites were also labeled by anti-lectin antibodies. Most of the labeled hepatocytes showed variable degrees of cell damage. Hepatocytes distantly localized from the parasites were also stained with the anti-lectin antibodies. Immunolabeling of tissue sections from different stages of the development of experimental amoebic liver abscess in hamsters showed inflammatory foci containing lectin-labeled trophozoites, hepatocytes, and sinusoidal and inflammatory cells. Lectin-containing hepatocytes had vacuolated cytoplasm with some nuclei with a condensed appearance. Damaged intestinal epithelium also was labeled with anti-lectin antibodies in a mouse model of intestinal amoebiasis. Electron microscopy of axenically cultured trophozoites using gold-labeled monoclonal and polyclonal anti-lectin antibody showed that plasma membrane, vacuole membranes and areas of cell cytosol were labeled. Higher deposits of gold particles in plasma membrane suggestive of cell secretion were observed. Our results demonstrated that Gal/GalNAc lectin was bound and captured by different target cells, and that host cells containing the lectin showed signs of cell damage. The contribution of lectin transfer to host cells in adherence and cell injury remains to be determined.

Entamoeba histolytica: sequence conservation of the Gal/GalNAc lectin from clinical isolates.

The Gal/GalNAc lectin gene of Entamoeba histolytica is a major amebic virulence protein responsible for interaction with host tissues. We investigated sequence differences in the Gal/GalNAc lectin heavy subunit in three isolates from Bangladesh and one isolate from Georgia, each of which was determined to be genetically distinct by SREHP AluI digestion. Interestingly, we observed only slight genetic diversity in the lectin gene as compared with the HM1:IMSS laboratory strain, originally a clinical isolate from Mexico. Genetic conservation of the Gal/GalNAc lectin between isolates may reflect that the lectin is under strong functional selection or possibly, that E. histolytica is a clonal population. Sequence conservation of the lectin indicates that immune responses against it should be cross-protective.

Sequence-, tissue-, and delivery-specific targeting of RNA during post-transcriptional gene silencing.

Transgenic Nicotiana benthamiana plants expressing an untranslatable version of the coat protein (CP) gene from the Tamarillo mosaic virus (TaMV) were either resistant to TaMV infection or recovered from infection. These phenotypes were the result of a post-transcriptional gene silencing (PTGS) mechanism that targeted TaMV-CP sequences for degradation. The TaMV-CP sequences were degraded when present in the wild-type TaMV potyvirus, in transgene mRNA, or in chimeric viral vectors based on White clover mosaic virus. The more efficiently targeted region was mapped to a 134-nt segment. Differences were observed in the efficiency of targeting during cell-to-cell and long-distance movement of the chimeric viruses. However, the TaMV-CP sequences do not appear to be targeted for degradation when delivered by biolistics.