Development of an improved medium for the isolation of 'Haemophilus somnus'

A medium containing lincomycin (3 μg/ml), cycloheximide (100 μg/ml) and chloral hydrate (0–1 %) was superior to all others examined for the isolation of 'Haemophilus somnus' from material contaminated with Proteus species.     

Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

Targeting of specific domains of diphtheria toxin by site-directed antibodies.

Antibodies highly selective for two functionally distinct regions of diphtheria toxin (DTx) were prepared using synthetic peptide conjugates as immunogens. Three peptides were selected for synthesis: sequence DTx141-157 on fragment A, which contains the putative protein elongation factor (EF-2) ADP-ribosyltransferase site; DTx224-237 on fragment B, selected on the basis of forming a predicted surface loop; and DTx513-526 on fragment B, forming a part of the region containing the putative receptor binding domain. All of the anti-peptide antibodies recognized the corresponding peptide, and also reacted with the toxin, specifically with the fragment containing the sequence against which they were raised, confirming the utility of this approach in generating fragment-specific antibodies. The anti-peptide antibody with the highest binding titre both to the peptide and to the native toxin was the one prepared against the sequence with the highest surface and loop likelihood indices of the three peptides selected. The similarity of the reactivity profiles with peptide and native and denatured toxin is consistent with the prediction that the region selected occurs in a surface loop and that the structure of the peptide is similar to the conformation of this region in the native protein. The epitopes for two of the anti-peptide antibodies were mapped. The results indicated that even though the antisera were raised to peptides containing 14 amino acids (aa) they were directed predominantly against a narrow region within the peptide, consisting of only 5-6 aa residues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and properties of a phage lytic for non-smooth Brucella organisms

A phage for non-smooth cells of Brucella abortus was isolated from a mixture of three brucella-phages incubated with rough brucella cells in the presence of N-methyl-N'-nitro-N-nitrosoguanidine. It did not lyse smooth cells of Br. abortus strains nor those of other Brucella species, but during the course of its replication in rough organisms a small proportion of phage mutants were produced which were similar in properties to smooth-specific phages. The rough-specific phage, R, itself resembled Weybridge phage in its morphological and serological properties. Phage R was found to contain DNA and segregated into high and low density fractions, both with plaque-forming activity, on ultra-centrifugation in CsSO4 gradients. Unlike the smooth-specific phages it attached to heat-labile receptors present on non-smooth brucella cells.     

Serological biomarker for assessing human exposure to Aedes mosquito bites during a randomized vector control intervention trial in northeastern Thailand

BackgroundAedes mosquitoes are vectors for several major arboviruses of public health concern including dengue viruses. The relationships between Aedes infestation and disease transmission are complex wherein the epidemiological dynamics can be difficult to discern because of a lack of robust and sensitive indicators for predicting transmission risk. This study investigates the use of anti-Aedes saliva antibodies as a serological biomarker for Aedes mosquito bites to assess small scale variations in adult Aedes density and dengue virus (DENV) transmission risk in northeastern Thailand. Individual characteristics, behaviors/occupation and socio-demographics, climatic and epidemiological risk factors associated with human-mosquito exposure are also addressed.MethodsThe study was conducted within a randomized clustered control trial in Roi Et and Khon Kaen provinces over a consecutive 19 months period. Thirty-six (36) clusters were selected, each of ten houses. Serological and entomological surveys were conducted in all houses every four months and monthly in three sentinel households per cluster between September 2017 and April 2019 for blood spot collections and recording concurrent immature and adult Aedes indices. Additionally, the human exposure to Aedes mosquito bites (i.e., Mosquito Exposure Index or MEI) was estimated by ELISA measuring levels of human antibody response to the specific Nterm-34 kDa salivary antigen. The relationships between the MEI, vector infestation indices (adult and immature stages) and vector DENV infection were evaluated using a two-level (house and individual levels) mixed model analysis with one-month lag autoregressive correlation.ResultsThere was a strong positive relationship between the MEI and adult Aedes (indoor and outdoor) density. Individuals from households with a medium mosquito density (mean difference: 0.091, p<0.001) and households with a high mosquito density (mean difference: 0.131, p<0.001) had higher MEI’s compared to individuals from households without Aedes. On a similar trend, individuals from households with a low, medium or high indoor Aedes densities (mean difference: 0.021, p<0.007, 0.053, p<0.0001 and 0.037, p<0.0001 for low, medium and high levels of infestation, respectively) had higher MEI than individuals from houses without indoor Aedes. The MEI was driven by individual characteristics, such as gender, age and occupation/behaviors, and varied according to climatic, seasonal factors and vector control intervention (p<0.05). Nevertheless, the study did not demonstrate a clear correlation between MEI and the presence of DENV-infected Aedes.ConclusionThis study represents an important step toward the validation of the specific IgG response to the Aedes salivary peptide Nterm-34kDa as a proxy measure for Aedes infestation levels and human-mosquito exposure risk in a dengue endemic setting. The use of the IgG response to the Nterm-34 kDa peptide as a viable diagnostic tool for estimating dengue transmission requires further investigations and validation in other geographical and transmission settings.     

The role of snail aestivation in transmission of schistosomiasis in changing climatic conditions

Schistosomiasis vector snails are subjected to extreme seasonal changes, particularly in ephemeral rivers and lentic waterbodies. In the tropics, aestivation is one of the adaptive strategies for survival and is used by snails in times of extremely high temperatures and desiccation. Aestivation therefore plays an important role in maintaining the transmission of schistosomiasis. This review assesses the possible impacts of climate change on the temporal and spatial distribution of schistosomiasis-transmitting snails with special emphasis on aestivation, and discusses the effect of schistosome infection on aestivation ability. The impacts of parasite development on snails, as well as physiological changes, are discussed with reference to schistosomiasis transmission. This review shows that schistosome-infected snails have lower survival rates during aestivation, and that those that survive manage to get rid of the infection. In general, snail aestivation ability is poor and survival chances diminish with time. Longer dry periods result in fewer, as well as uninfected, snails. However, the ability of the surviving snails to repopulate the habitats is high.     

Attenuation of pattern recognition receptor signaling is mediated by a MAP kinase kinase kinase

Pattern recognition receptors (PRRs) play a key role in plant and animal innate immunity. PRR binding of their cognate ligand triggers a signaling network and activates an immune response. Activation of PRR signaling must be controlled prior to ligand binding to prevent spurious signaling and immune activation. Flagellin perception in Arabidopsis through FLAGELLIN‐SENSITIVE 2 (FLS2) induces the activation of mitogen‐activated protein kinases (MAPKs) and immunity. However, the precise molecular mechanism that connects activated FLS2 to downstream MAPK cascades remains unknown. Here, we report the identification of a differentially phosphorylated MAP kinase kinase kinase that also interacts with FLS2. Using targeted proteomics and functional analysis, we show that MKKK7 negatively regulates flagellin‐triggered signaling and basal immunity and this requires phosphorylation of MKKK7 on specific serine residues. MKKK7 attenuates MPK6 activity and defense gene expression. Moreover, MKKK7 suppresses the reactive oxygen species burst downstream of FLS2, suggesting that MKKK7‐mediated attenuation of FLS2 signaling occurs through direct modulation of the FLS2 complex.