Genotyping of Human Lice Suggests Multiple Emergences of Body Lice from Local Head Louse Populations

Background

Genetic analyses of human lice have shown that the current taxonomic classification of head lice (Pediculus humanus capitis) and body lice (Pediculus humanus humanus) does not reflect their phylogenetic organization. Three phylotypes of head lice A, B and C exist but body lice have been observed only in phylotype A. Head and body lice have different behaviours and only the latter have been involved in outbreaks of infectious diseases including epidemic typhus, trench fever and louse borne recurrent fever. Recent studies suggest that body lice arose several times from head louse populations.

Methods and Findings

By introducing a new genotyping technique, sequencing variable intergenic spacers which were selected from louse genomic sequence, we were able to evaluate the genotypic distribution of 207 human lice. Sequence variation of two intergenic spacers, S2 and S5, discriminated the 207 lice into 148 genotypes and sequence variation of another two intergenic spacers, PM1 and PM2, discriminated 174 lice into 77 genotypes. Concatenation of the four intergenic spacers discriminated a panel of 97 lice into 96 genotypes. These intergenic spacer sequence types were relatively specific geographically, and enabled us to identify two clusters in France, one cluster in Central Africa (where a large body louse outbreak has been observed) and one cluster in Russia. Interestingly, head and body lice were not genetically differentiated.

Conclusions

We propose a hypothesis for the emergence of body lice, and suggest that humans with both low hygiene and head louse infestations provide an opportunity for head louse variants, able to ingest a larger blood meal (a required characteristic of body lice), to colonize clothing. If this hypothesis is ultimately supported, it would help to explain why poor human hygiene often coincides with outbreaks of body lice. Additionally, if head lice act as a reservoir for body lice, and that any social degradation in human populations may allow the formation of new populations of body lice, then head louse populations are potentially a greater threat to humans than previously assumed.     

The mitochondrial genome of the chimpanzee louse,Pediculus schaeffi: insights into the process of mitochondrial genome fragmentation in the blood-sucking lice of great apes

Background

Blood-sucking lice in the genera Pediculus and Pthirus are obligate ectoparasites of great apes. Unlike most bilateral animals, which have 37 mitochondrial (mt) genes on a single circular chromosome, the sucking lice of humans have extensively fragmented mt genomes. The head louse, Pediculus capitis, and the body louse, Pe. humanus, have their 37 mt genes on 20 minichromosomes. The pubic louse, Pthirus pubis, has its 34 mt genes known on 14 minichromosomes. To understand the process of mt genome fragmentation in the sucking lice of great apes, we sequenced the mt genome of the chimpanzee louse, Pe. schaeffi, and compared it with the three human lice.

Results

We identified all of the 37 mt genes typical of bilateral animals in the chimpanzee louse; these genes are on 18 types of minichromosomes. Seventeen of the 18 minichromosomes of the chimpanzee louse have the same gene content and gene arrangement as their counterparts in the human head louse and the human body louse. However, five genes, cob, trnS₁, trnN, trnE and trnM, which are on three minichromosomes in the human head louse and the human body louse, are together on one minichromosome in the chimpanzee louse.

Conclusions

Using the human pubic louse, Pt. pubis, as an outgroup for comparison, we infer that a single minichromosome has fragmented into three in the lineage leading to the human head louse and the human body louse since this lineage diverged from the chimpanzee louse ~6 million years ago. Our results provide insights into the process of mt genome fragmentation in the sucking lice in a relatively fine evolutionary scale.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1843-3) contains supplementary material, which is available to authorized users.     

Heteroplasmy in the Mitochondrial Genomes of Human Lice and Ticks Revealed by High Throughput Sequencing

The typical mitochondrial (mt) genomes of bilateral animals consist of 37 genes on a single circular chromosome. The mt genomes of the human body louse, Pediculus humanus, and the human head louse, Pediculus capitis, however, are extensively fragmented and contain 20 minichromosomes, with one to three genes on each minichromosome. Heteroplasmy, i.e. nucleotide polymorphisms in the mt genome within individuals, has been shown to be significantly higher in the mt cox1 gene of human lice than in humans and other animals that have the typical mt genomes. To understand whether the extent of heteroplasmy in human lice is associated with mt genome fragmentation, we sequenced the entire coding regions of all of the mt minichromosomes of six human body lice and six human head lice from Ethiopia, China and France with an Illumina HiSeq platform. For comparison, we also sequenced the entire coding regions of the mt genomes of seven species of ticks, which have the typical mitochondrial genome organization of bilateral animals. We found that the level of heteroplasmy varies significantly both among the human lice and among the ticks. The human lice from Ethiopia have significantly higher level of heteroplasmy than those from China and France (P_t<0.05). The tick, Amblyomma cajennense, has significantly higher level of heteroplasmy than other ticks (P_t<0.05). Our results indicate that heteroplasmy level can be substantially variable within a species and among closely related species, and does not appear to be determined by single factors such as genome fragmentation.     

Predicting Essential Metabolic Genome Content of Niche-Specific Enterobacterial Human Pathogens during Simulation of Host Environments

Microorganisms have evolved to occupy certain environmental niches, and the metabolic genes essential for growth in these locations are retained in the genomes. Many microorganisms inhabit niches located in the human body, sometimes causing disease, and may retain genes essential for growth in locations such as the bloodstream and urinary tract, or growth during intracellular invasion of the hosts’ macrophage cells. Strains of Escherichia coli (E. coli) and Salmonella spp. are thought to have evolved over 100 million years from a common ancestor, and now cause disease in specific niches within humans. Here we have used a genome scale metabolic model representing the pangenome of E. coli which contains all metabolic reactions encoded by genes from 16 E. coli genomes, and have simulated environmental conditions found in the human bloodstream, urinary tract, and macrophage to determine essential metabolic genes needed for growth in each location. We compared the predicted essential genes for three E. coli strains and one Salmonella strain that cause disease in each host environment, and determined that essential gene retention could be accurately predicted using this approach. This project demonstrated that simulating human body environments such as the bloodstream can successfully lead to accurate computational predictions of essential/important genes.     

Recent approaches for control of E. coli and respiratory complex in Middle East

This study was conducted on 100 one-day-old broiler chicks to evaluate the effect of Poulvac E. coli vaccine in reduction of clinical signs and complications after concurrent infectious bronchitis virus (variant 02) and virulent E. coli O78 challenges. The birds were evaluated for clinical signs, mortality for 7?days post-infection, PM lesion score, average body weight and serological evaluation. Re-isolation and RT-PCR for the challenging infectious bronchitis virus (IBV) variant 02 were conducted thereafter. The results showed that the Poulvac E. coli at one-day old chicks in the presence of co-infection with virulent E. coli and IBV variant 02 provides better body weight gain at 35?days than the other groups. The challenge with IBV variant 02 alone in non-vaccinated birds doesn’t give any mortality; this indicated that the severity of IBV variant 02 increased by the presence of co-infection with Avian Pathogenic E. coli (APEc). The mortality percentage associated with both E. coli and IBV variant 02 infections in the none vaccinated group by Poulvac E. coli was 25% while this percentage was 10% of the vaccinated group. The Poulvac E. coli is not negatively affecting the immune response against different concurrent viral vaccines like Infectious bursal disease (IBD), and moreover, it improves the immune response against some others like Newcastle disease virus (NDV), Avian Influenza (AI) H5 and IBV.     

Phenotypic and Genotypic Characterization of Avian Escherichia coli O86:K61 Isolates Possessing a Gamma-Like Intimin

Escherichia coli O86:K61 has long been associated with outbreaks of infantile diarrhea in humans and with diarrheal disease in many animal species. Studies in the late 1990s identified E. coli O86:K61 as the cause of mortality in a variety of wild birds, and in this study, 34 E. coli O86:K61 isolates were examined. All of the isolates were nonmotile, but most elaborated at least two morphologically distinct surface appendages that were confirmed to be type 1 and curli fimbriae. Thirty-three isolates were positive for the eaeA gene encoding a gamma type of intimin. No phenotypic or genotypic evidence was obtained for elaboration of Shiga-like toxins, but most isolates possessed the gene coding for the cytolethal distending toxin. Five isolates were selected for adherence assays performed with tissue explants and HEp-2 cells, and four of these strains produced attaching and effacing lesions on HEp-2 cells and invaded the cells, as determined by transmission electron microscopy. Two of the five isolates were inoculated orally into 1-day-old specific-pathogen-free chicks, and both of these isolates colonized, invaded, and persisted well in this model. Neither isolate produced attaching and effacing lesions in chicks, although some pathology was evident in the alimentary tract. No deaths were recorded in inoculated chicks. These findings are discussed in light of the possibility that wild birds are potential zoonotic reservoirs of attaching and effacing E. coli.     

Identification and validation of reference genes for real time PCR expression studies in heads of nurse honeybees

The primary aim of this study was to identify reference genes and workers of particular role and ages that would be suitable for exploring genetic/epigenetic variations in constitutive expression of a gene encoding antimicrobial peptide defensin1 in worker heads using real-time PCR. This peptide is an integral component of larval food and honey and has potential to act against some brood pathogens. Expression levels of distinct genes may vary in worker heads due to genetic factors, age of bee, and particular role of a worker that depends on its age or colony needs. Prerequisite for exploring the variations in defensin1 expression was therefore to identify such workers in which correlated expression of defensin1 and suitable reference genes occurs. Selection process was done by carefully designed quantitative real-time PCR procedure in two colonies showing different age-related division of labor. Expression of ten candidate reference genes, defensin1 and amylase, as a marker of forager bees, was assessed in pooled head samples of workers aged 2 to 30 days. Correlated and moreover stable expression of defensin1 and six candidate genes was detected in nursing bees in both colonies. The suitable reference genes were therefore selected on the basis of their expression stability. This was evaluated by geNorm and NormFinder algorithms in pooled head samples and through plotted Cq data in head samples of individual nurse bees. As the best reference genes were selected: psa1, tctp1, cyclophilin, gapdh and mrjp4 (in this order). They are suitable for aforementioned defensin1 expression studies and also for studies of other genes expressed in heads of nurses. In addition, an amylase expression-based procedure for reliable distinguishing nurses from foragers was elaborated.     

Transcriptome identification of putative genes involved in protein catabolism and innate immune response in human body louse (Pediculicidae: Pediculus humanus)

Genomics information relating to human body lice is surprisingly scarce, and this has constrained studies of their physiology, immunology and vector biology. To identify novel body louse genes, we used engorged adult lice to generate a cDNA library. Initially, 1152 clones were screened for inserts, edited for removal of vector sequences and base pairs of poor quality, and viewed for splicing variations, gene families and polymorphism. Computational methods identified 506 inferred open reading frames including the first predicted louse defensin. The inferred defensin aligns well with other insect defensins and has highly conserved cysteine residues, as are known for other defensin sequences. Two cysteine and five serine proteinases were categorized according to their inferred catalytic sites. We also discovered seven putative ubiquitin-pathway genes and four iron metabolizing deduced enzymes. Finally, glutathione-S-transferases and cytochrome P450 genes were among the detoxification enzymes found. Results from this first systematic effort to discover human body louse genes should promote further studies in Phthiraptera and lice.     

Quantitative studies on trypanosomes in tsetse tissue culture

Tissues from pupae of Glossina morsitans of various ages were cultured in modified Trager's medium. Cellular outgrowths were produced from explants of proventriculus, brain, and imaginal body wall and large vesicles were extruded from pieces of midgut of young pupae. Complete alimentary tract from older pupae displayed rhythmic contractions for up to 3 weeks. When Trypanosoma brucei and T. congolense in mouse blood were added to hanging drop cultures of tsetse tissues and incubated at 28 C, the organisms multiplied and changed into forms morphologically similar to those found in the tsetse fly midgut. The trypanosomes were maintained for 30 days by serial passage at 5-day intervals. The growth of T. brucei in the presence of different pupal tissues was studied. Of all the tissues tested the complete alimentary tract from pupae older than 21 days gave the best results. Growth also occurred when the trypanosomes were separated from the insect tissue by a semipermeable membrane. The trypanosomes failed to grow in (a) culture medium alone, (b) media containing extracts of alimentary canal and (c) medium in which alimentary tract had been cultured for 3 or 4 days.     

A glyco-competitive assay to demonstrate the stochasticity of fate decisions in Escherichia coli

Interaction bacteria-gut, via glycan associations, contribute to the selection of microbial communities along the gastrointestinal tract, influencing cancer development. The mechanism causing microbiome alterations is unknown, while this understanding would be pivotal to identify medical therapies. The molecular associations between Escherichia coli bacteria and glucose, both in solution and immobilized at the surface, were studied showing the dependence of E. coli glucose binding on the sugar form. Classical kinetic models were used to derive the reaction equilibrium and adsorption constants, 8 mM⁻¹ and 1 (cell/mL)⁻¹ and to explain the uptake. E. coli preferred the free glucose, whereas in a deprived environment, the anchored glucose became the major source of carbon for the bacteria. A stochastic algorithm disclosed that after initial transient, E. coli privileged the anchored glucose rather than the free sugar, independently on the concentration. The biochemical approach alone failed to describe the effective behavior of the cells and that several parameters can affect the behavior of the bacteria. From this result, more sophisticated models of the destruction of the gut barrier can be derived, such as the mechanism whereby E. coli can switch the immune system on and off to cause cancer and its metastasis.     

Cuterebra buccata: immune response in myiasis of domestic rabbits

Naturally infected rabbits (Oryctolagus) were used to define further the nature of the immune response in myiasis due to Cuterebra buccata. Third instar larvae were dissected into four fractions; (1) alimentary tract with attached organs, (2) hemolymph, (3) fat body with tracheae, and (4) cuticle with attached muscles. The antigens provoking immune phenomena in naturally infected rabbits were found to reside in the alimentary tract and hemolymph fractions only. All rabbits which were skin tested were found to exhibit delayed hypersensitivity and to have serum precipitins with specificity against these antigens. Passive cutaneous anaphylaxis activity was demonstrated only in sera from rabbits also exhibiting reaginic and/or Arthus-type skin hypersensitivities. With the use of immunoelectrophoresis four separate antigens were demonstrated in alimentary tract fractions. Larval dissections revealed the alimentary tract to be filled with cellular elements of rabbit whole blood. The immunologic findings are discussed in relation to this newly recognized feeding pattern and it is proposed that sensitization of the host occurs as a consequence of exogenous larval secretions injected at the time of feeding.     

Complex environmental drivers of immunity and resistance in malaria mosquitoes

Considerable research effort has been directed at understanding the genetic and molecular basis of mosquito innate immune mechanisms. Whether environmental factors interact with these mechanisms to shape overall resistance remains largely unexplored. Here, we examine how changes in mean ambient temperature, diurnal temperature fluctuation and time of day of infection affected the immunity and resistance of Anopheles stephensi to infection with Escherichia coli. We used quantitative PCR to estimate the gene expression of three immune genes in response to challenge with heat-killed E. coli. We also infected mosquitoes with live E. coli and ran bacterial growth assays to quantify host resistance. Both mosquito immune parameters and resistance were directly affected by mean temperature, diurnal temperature fluctuation and time of day of infection. Furthermore, there was a suite of complex two- and three-way interactions yielding idiosyncratic phenotypic variation under different environmental conditions. The results demonstrate mosquito immunity and resistance to be strongly influenced by a complex interplay of environmental variables, challenging the interpretation of the very many mosquito immune studies conducted under standard laboratory conditions.