Molecular Evidence for the Presence of Rickettsia Felis in the Feces of Wild-living African Apes

Background

Rickettsia felis is a common emerging pathogen detected in mosquitoes in sub-Saharan Africa. We hypothesized that, as with malaria, great apes may be exposed to the infectious bite of infected mosquitoes and release R. felis DNA in their feces.

Methods

We conducted a study of 17 forest sites in Central Africa, testing 1,028 fecal samples from 313 chimpanzees, 430 gorillas and 285 bonobos. The presence of rickettsial DNA was investigated by specific quantitative real-time PCR. Positive results were confirmed by a second PCR using primers and a probe targeting a specific gene for R. felis. All positive samples were sequenced.

Results

Overall, 113 samples (11%) were positive for the Rickettsia-specific gltA gene, including 25 (22%) that were positive for R. felis. The citrate synthase (gltA) sequence and outer membrane protein A (ompA) sequence analysis indicated 99% identity at the nucleotide level to R. felis. The 88 other samples (78%) were negative using R. felis-specific qPCR and were compatible with R. felis-like organisms.

Conclusion

For the first time, we detected R. felis in wild-living ape feces. This non invasive detection of human pathogens in endangered species opens up new possibilities in the molecular epidemiology and evolutionary analysis of infectious diseases, beside HIV and malaria.     

Prevalence and infection load dynamics of Rickettsia felis in actively feeding cat fleas

Background

Rickettsia felis is a flea-associated rickettsial pathogen recurrently identified in both colonized and wild-caught cat fleas, Ctenocephalides felis. We hypothesized that within colonized fleas, the intimate relationship between R. felis and C. felis allows for the coordination of rickettsial replication and metabolically active periods during flea bloodmeal acquisition and oogenesis.

Methodology/Principal Findings

A quantitative real-time PCR assay was developed to quantify R. felis in actively feeding R. felis-infected fleas. In three separate trials, fleas were allowed to feed on cats, and a mean of 3.9×10⁶ R. felis 17-kDa gene copies was detected for each flea. A distinct R. felis infection pattern was not observed in fleas during nine consecutive days of bloodfeeding. However, an inverse correlation between the prevalence of R. felis-infection, which ranged from 96% in Trial 1 to 35% in Trial 3, and the R. felis-infection load in individual fleas was identified. Expression of R. felis-infection load as a ratio of R. felis/C. felis genes confirmed that fleas in Trial 3 had significantly greater rickettsial loads than those in Trial 1.

Conclusion/Significance

Examining rickettsial infection dynamics in the flea vector will further elucidate the intimate relationship between R. felis and C. felis, and facilitate a more accurate understanding of the ecology and epidemiology of R. felis transmission in nature.     

Dissemination of Spotted Fever Rickettsia Agents in Europe by Migrating Birds

Migratory birds are known to play a role as long-distance vectors for many microorganisms. To investigate whether this is true of rickettsial agents as well, we characterized tick infestation and gathered ticks from 13,260 migratory passerine birds in Sweden. A total of 1127 Ixodes spp. ticks were removed from these birds and the extracted DNA from 957 of them was available for analyses. The DNA was assayed for detection of Rickettsia spp. using real-time PCR, followed by DNA sequencing for species identification. Rickettsia spp. organisms were detected in 108 (11.3%) of the ticks. Rickettsia helvetica, a spotted fever rickettsia associated with human infections, was predominant among the PCR-positive samples. In 9 (0.8%) of the ticks, the partial sequences of 17kDa and ompB genes showed the greatest similarity to Rickettsia monacensis, an etiologic agent of Mediterranean spotted fever-like illness, previously described in southern Europe as well as to the Rickettsia sp.IrITA3 strain. For 15 (1.4%) of the ticks, the 17kDa, ompB, gltA and ompA genes showed the greatest similarity to Rickettsia sp. strain Davousti, Rickettsia japonica and Rickettsia heilongjiangensis, all closely phylogenetically related, the former previously found in Amblyomma tholloni ticks in Africa and previously not detected in Ixodes spp. ticks. The infestation prevalence of ticks infected with rickettsial organisms was four times higher among ground foraging birds than among other bird species, but the two groups were equally competent in transmitting Rickettsia species. The birds did not seem to serve as reservoir hosts for Rickettsia spp., but in one case it seems likely that the bird was rickettsiemic and that the ticks had acquired the bacteria from the blood of the bird. In conclusion, migratory passerine birds host epidemiologically important vector ticks and Rickettsia species and contribute to the geographic distribution of spotted fever rickettsial agents and their diseases.     

Relative Sensitivity of Conventional and Real-Time PCR Assays for Detection of SFG Rickettsia in Blood and Tissue Samples from Laboratory Animals

Studies on the natural transmission cycles of zoonotic pathogens and the reservoir competence of vertebrate hosts require methods for reliable diagnosis of infection in wild and laboratory animals. Several PCR-based applications have been developed for detection of infections caused by Spotted Fever group Rickettsia spp. in a variety of animal tissues. These assays are being widely used by researchers, but they differ in their sensitivity and reliability. We compared the sensitivity of five previously published conventional PCR assays and one SYBR green-based real-time PCR assay for the detection of rickettsial DNA in blood and tissue samples from Rickettsia- infected laboratory animals (n = 87). The real-time PCR, which detected rickettsial DNA in 37.9% of samples, was the most sensitive. The next best were the semi-nested ompA assay and rpoB conventional PCR, which detected as positive 18.4% and 14.9% samples respectively. Conventional assays targeting ompB, gltA and hrtA genes have been the least sensitive. Therefore, we recommend the SYBR green-based real-time PCR as a tool for the detection of rickettsial DNA in animal samples due to its higher sensitivity when compared to more traditional assays.     

Rickettsia felis Infection in a Common Household Insect Pest,Liposcelis bostrychophila (Psocoptera: Liposcelidae)

Many species of Rickettsia are well-known mammalian pathogens transmitted by blood-feeding arthropods. However, molecular surveys are continually uncovering novel Rickettsia species, often in unexpected hosts, including many arthropods that do not feed on blood. This study reports a systematic molecular characterization of a Rickettsia infecting the psocid Liposcelis bostrychophila (Psocoptera: Liposcelidae), a common and cosmopolitan household pest. Surprisingly, the psocid Rickettsia is shown to be Rickettsia felis, a human pathogen transmitted by fleas that causes serious morbidity and occasional mortality. The plasmid from the psocid R. felis was sequenced and was found to be virtually identical to the one in R. felis from fleas. As Liposcelis insects are often intimately associated with humans and other vertebrates, it is speculated that they acquired R. felis from fleas. Whether the R. felis in psocids causes disease in vertebrates is not known and warrants further study.Many species of Rickettsia are well-known mammalian pathogens that are transmitted by blood-feeding arthropods via bites or feces and can cause mild to fatal diseases in humans (33). Some species are also considered potential bioterrorism agents (4). Most Rickettsia research has focused on pathogens that are found in two closely related species groups, the typhus and spotted fever groups, such as Rickettsia prowazekii, Rickettsia rickettsii, and Rickettsia typhi, the causal agents of epidemic typhus, Rocky Mountain spotted fever, and murine typhus, respectively (3, 4, 33). However, recent surveys suggest that Rickettsia bacteria are much more widespread than previously suspected and that they are being detected in novel hosts, the vast majority of which are arthropods, including many that do not feed on blood (29, 45).The number of new rickettsial species that cause diseases in humans is rapidly increasing (33). One such species that has been generating much interest in recent years is Rickettsia felis, the causative agent of a murine typhus-like disease (1, 2, 13, 16, 17, 28, 44). The disease is often unrecognized, and even though it is considered clinically mild, it can cause severe illness and death in older patients and in cases of delayed diagnosis (2). R. felis was identified only in 1990 (1) and has since been found worldwide in fleas, where it is maintained transovarially and can reach high infection rates (e.g., 86% to 94% in cat fleas) (2, 3, 44), as well as in ticks and mites (34). While experimental infections have confirmed that R. felis is transmitted to vertebrate hosts via blood feeding and that R. felis occurs in an infectious extracellular state (39), it is not known whether transmission can also occur through contamination of broken skin by infected vector feces, as in R. typhi (3, 34).A number of features distinguish R. felis from species in both the typhus and spotted fever groups. Lately, it has been proposed that R. felis be in its own group, allied with Rickettsia akari and Rickettsia australis, the causal agents of rickettsial pox and Queensland tick typhus, respectively, and a number of recently discovered strains infecting insects that do not feed on blood (16, 17, 29, 45). Moreover, R. felis was the first Rickettsia species shown to have a plasmid (28). While plasmids now appear to be quite widespread in the genus, the R. felis plasmid stands out with respect to its relatively large size and distinctive gene content (5, 6, 9, 14, 17).This study reports that a common and cosmopolitan insect, the psocid Liposcelis bostrychophila (Psocoptera: Liposcelidae) harbors R. felis. Liposcelids are the closest free-living relatives of parasitic lice (19) and are well-known for their close proximity to humans, particularly as pests in houses and grain storage facilities (8, 41). Through 16S rRNA gene sequencing, L. bostrychophila was recently shown to harbor a strain of Rickettsia (29, 30, 42). A systematic molecular characterization of this Rickettsia was conducted, demonstrating that it is authentic R. felis. Furthermore, the psocid symbiont plasmid was sequenced and was shown to be virtually identical to the plasmid from R. felis that infects cat fleas.     

Propagation of Arthropod-Borne Rickettsia spp. in Two Mosquito Cell Lines

Rickettsiae are obligate intracellular alphaproteobacteria that include pathogenic species in the spotted fever, typhus, and transitional groups. The development of a standardized cell line in which diverse rickettsiae can be grown and compared would be highly advantageous to investigate the differences among and between pathogenic and nonpathogenic species of rickettsiae. Although several rickettsial species have been grown in tick cells, tick cells are more difficult to maintain and they grow more slowly than insect cells. Rickettsia-permissive arthropod cell lines that can be passaged rapidly are highly desirable for studies on arthropod-Rickettsia interactions. We used two cell lines (Aedes albopictus cell line Aa23 and Anopheles gambiae cell line Sua5B) that have not been used previously for the purpose of rickettsial propagation. We optimized the culture conditions to propagate one transitional-group rickettsial species (Rickettsia felis) and two spotted-fever-group rickettsial species (R. montanensis and R. peacockii) in each cell line. Both cell lines allowed the stable propagation of rickettsiae by weekly passaging regimens. Stable infections were confirmed by PCR, restriction digestion of rompA, sequencing, and the direct observation of bacteria by fluorescence in situ hybridization. These cell lines not only supported rickettsial growth but were also permissive toward the most fastidious species of the three, R. peacockii. The permissive nature of these cell lines suggests that they may potentially be used to isolate novel rickettsiae or other intracellular bacteria. Our results have important implications for the in vitro maintenance of uncultured rickettsiae, as well as providing insights into Rickettsia-arthropod interactions.     

Different mutation patterns of atovaquone resistance to Plasmodium falciparum in vitro and in vivo : rapid detection of codon 268 polymorphisms in the cytochrome b as potential in vivo resistance marker

Background

Anopheles gambiae is the main vector of Plasmodium falciparum in Africa. The mosquito midgut constitutes a barrier that the parasite must cross if it is to develop and be transmitted. Despite the central role of the mosquito midgut in the host/parasite interaction, little is known about its protein composition. Characterisation of An. gambiae midgut proteins may identify the proteins that render An. gambiae receptive to the malaria parasite.

Methods

We carried out two-dimensional gel electrophoresis of An. gambiae midgut proteins and compared protein profiles for midguts from males, sugar-fed females and females fed on human blood.

Results

Very few differences were detected between male and female mosquitoes for the approximately 375 silver-stained proteins. Male midguts contained ten proteins not detected in sugar-fed or blood-fed females, which are therefore probably involved in male-specific functions; conversely, female midguts contained twenty-three proteins absent from male midguts. Eight of these proteins were specific to sugar-fed females, and another ten, to blood-fed females.

Conclusion

Mass spectrometry analysis of the proteins found only in blood-fed female midguts, together with data from the recent sequencing of the An. gambiae genome, should make it possible to determine the role of these proteins in blood digestion or parasite receptivity.     

Wide Dispersal and Possible Multiple Origins of Low-Copy-Number Plasmids in Rickettsia Species Associated with Blood-Feeding Arthropods

Plasmids are mobile genetic elements of bacteria that can impart important adaptive traits, such as increased virulence or antibiotic resistance. We report the existence of plasmids in Rickettsia (Rickettsiales; Rickettsiaceae) species, including Rickettsia akari, “Candidatus Rickettsia amblyommii,” R. bellii, R. rhipicephali, and REIS, the rickettsial endosymbiont of Ixodes scapularis. All of the rickettsiae were isolated from humans or North and South American ticks. R. parkeri isolates from both continents did not possess plasmids. We have now demonstrated plasmids in nearly all Rickettsia species that we have surveyed from three continents, which represent three of the four major proposed phylogenetic groups associated with blood-feeding arthropods. Gel-based evidence consistent with the existence of multiple plasmids in some species was confirmed by cloning plasmids with very different sequences from each of two “Ca. Rickettsia amblyommii” isolates. Phylogenetic analysis of rickettsial ParA plasmid partitioning proteins indicated multiple parA gene origins and plasmid incompatibility groups, consistent with possible multiple plasmid origins. Phylogenetic analysis of potentially host-adaptive rickettsial small heat shock proteins showed that hsp2 genes were plasmid specific and that hsp1 genes, found only on plasmids of “Ca. Rickettsia amblyommii,” R. felis, R. monacensis, and R. peacockii, were probably acquired independently of the hsp2 genes. Plasmid copy numbers in seven Rickettsia species ranged from 2.4 to 9.2 per chromosomal equivalent, as determined by real-time quantitative PCR. Plasmids may be of significance in rickettsial evolution and epidemiology by conferring genetic plasticity and host-adaptive traits via horizontal gene transfer that counteracts the reductive genome evolution typical of obligate intracellular bacteria.The alphaproteobacteria of the genus Rickettsia (Rickettsiales; Rickettsiaceae) have undergone the reductive genome evolution typical of obligate intracellular bacteria, resulting in A/T-rich genomes (1.1 × 10⁶ to 1.5 × 10⁶ bp) with a high content of pseudogenes undergoing elimination (3, 10, 20, 26). Initial sequencing of rickettsial genomes focused on the important arthropod-borne pathogens Rickettsia prowazekii, Rickettsia conorii, and Rickettsia typhi and appeared to confirm the prevailing belief that plasmids were absent and transposons were rare among Rickettsia spp. (2, 28, 39, 44). As mobile genetic elements in bacteria, plasmids and transposons drive horizontal gene transfer (HGT) and the acquisition of virulence determinants and environmental adaptive traits (30, 43, 60, 70). Subsequent sequencing of the Rickettsia felis genome revealed the surprising presence of abundant transposase paralogs and the 63-kbp pRF plasmid, with 68 open reading frames (ORFs) encoding predicted proteins, as well as a 39-kbp deletion form, pRFδ (45). Although pRF was suggested to be conjugative, it was initially thought to be unique among the rickettsiae, a reasonable inference given that plasmids are uncommon among the reduced genomes of obligate intracellular bacteria and were previously unknown in the Rickettsiales (3, 4, 13). However, a phylogenetic analysis implied an origin for pRF in ancestral rickettsiae and the possible existence of other rickettsial plasmids (28), which was soon confirmed by the cloning of the 23.5-kbp pRM plasmid from Rickettsia monacensis (6). Some of the 23 ORFs on pRM had close pRF homologs, and both plasmids carried transposon genes and the molecular footprints of transposition events associated with HGT from other bacterial taxa.The discoveries of pRF and pRM made obsolete the long-held dogma that plasmids were not present in members of the genus Rickettsia and implied a source of unexpected genetic diversity in the reduced rickettsial genomes, particularly if potentially conjugative plasmids carrying transposon genes proved to be common among members of the genus. That hypothesis gained credence when pulsed-field gel electrophoresis (PFGE) and Southern blot surveys (7) using plasmid gene-specific probes demonstrated plasmids in Rickettsia helvetica, “Candidatus Rickettsia hoogstraalii” (38), and Rickettsia massiliae and possible multiple plasmids in “Candidatus Rickettsia amblyommii” (71) isolates. The same study demonstrated the loss of a plasmid in the nonpathogenic species Rickettsia peacockii during long-term serial passage in cultured cells and the absence of a plasmid in Rickettsia montanensis M5/6, an isolate with a long laboratory passage history. Genome sequencing of R. massiliae and Rickettsia africae revealed the 15.3-kbp pRMA and 12.4-kbp pRAF sequences, with 12 and 11 ORFs, respectively, that were more similar to those of pRF than to those of pRM (11, 24).The absence of plasmids in R. montanensis and important Rickettsia pathogens maintained as laboratory isolates has left unresolved the question of the true extent of plasmid distribution among Rickettsia spp. Until recently, the genus was thought to consist of closely related species, known chiefly as typhus and spotted fever pathogens transmitted by lice, fleas, mites, and ticks (31). It is now apparent that many, and possibly most, Rickettsia spp. inhabit a diverse range of arthropods that do not feed on blood, as well as leeches, helminths, crustaceans, and protozoans, suggesting an ancient and complex evolutionary history (54). A multigene phylogenetic analysis of the Rickettsiales resulted in a “molecular clock” which indicated that the order arose from a presumably free-living ancestor and then adapted to intracellular growth during the appearance of metazoan phyla in the Cambrian explosion (76). A transition to a primary association with arthropods followed during the Ordovician and Silurian periods. The genus Rickettsia arose approximately 150 million years ago and evolved into several clades, including the early-diverging hydra and torix lineages associated with leeches and protozoans. A rapid radiation occurred about 50 million years ago in the arthropod-associated lineages (76).Whole-genome sequencing has led to a revision of phylogenetic relationships among Rickettsia spp. associated with blood-feeding arthropods (10, 26, 28). A newly defined ancestral group (AG) contains the earliest-diverging species, Rickettsia bellii and Rickettsia canadensis, while R. prowazekii and R. typhi, transmitted by lice and fleas, respectively, constitute the typhus group (TG). A proposed transitional group (TRG), consisting of the mite-borne Rickettsia akari, the flea-borne R. felis, and the tick-borne Rickettsia australis, bridges the genotypic and phenotypic differences between the TG and the much larger spotted fever group (SFG), consisting of tick-borne rickettsiae (28). However, some presumptive SFG rickettsiae remain poorly characterized and are of uncertain phylogenetic status, while the accumulation of genomic data from rickettsiae found in a diverse range of invertebrate hosts may have profound impacts on the currently understood phylogeny of rickettsiae associated with blood-feeding arthropods. For example, it appears that the above AG and TRG species have many close relatives in insects (76). Despite the recent phylogenomic advances, the genetic and host-adaptive mechanisms underlying the evolution of arthropod-transmitted pathogens of vertebrates from ancestral Rickettsia spp., including any possible role of plasmids, remain poorly understood.In this report, we have taken advantage of recent isolations of rickettsiae from North and South America to conclusively demonstrate that low-copy-number plasmids are indeed common in low-passage isolates of AG, TRG, and SFG rickettsiae. The only exceptions were multiple isolates of R. parkeri, obtained from ticks and human eschar biopsy specimens and newly recognized as a mildly pathogenic SFG rickettsia (49, 50, 52, 79), and the previously characterized species R. montanensis (7). We confirmed that some Rickettsia isolates harbor more than one plasmid by cloning and sequencing multiple plasmids from “Ca. Rickettsia amblyommii” isolates AaR/SC and Ac/Pa, and we obtained PCR- and gel-based evidence that supported genome sequence evidence for the existence of multiple plasmids in REIS, the rickettsial endosymbiont of Ixodes scapularis. Phylogenetic analysis provided strong evidence for multiple plasmid incompatibility groups and possible multiple origins of plasmid-carried parA genes in the genus Rickettsia. Other than genes encoding plasmid replication initiation and partitioning proteins, the newly sequenced “Ca. Rickettsia amblyommii” plasmids resembled the previously sequenced rickettsial plasmids in sharing limited similarities in coding capacity (6, 7, 22). However, we have previously drawn attention to the presence of hsp genes, encoding α-crystalline small heat shock proteins, as a conserved feature of most rickettsial plasmids that may play a role in host adaptation (7). Phylogenetic analysis indicated that the hsp2 genes were plasmid specific, while the hsp1 genes found on four rickettsial plasmids may have been acquired by a chromosome-to-plasmid transfer event in a TRG-like species.     

Rickettsia raoultii,the predominant Rickettsia found in Dermacentor silvarum ticks in China–Russia border areas

Since the year 2000, clinical patterns resembling tick-borne rickettsioses have been noticed in China–Russia border areas. Epidemiological data regarding species of the aetiological agent, tick vector prevalence and distribution as well as incidence of human cases in the areas are still sparse to date. In order to identify Rickettsia species occurring in the areas, we investigated Dermacentor silvarum collected in the selected areas. Rickettsia raoultii was the predominant Rickettsia found in D. silvarum evident with ompA, ompB, gltA and 17 kDa protein genes. The Rickettsia prevalence in D. silvarum appeared to be 32.25 % with no sex difference. The results extend the common knowledge about the geographic distribution of R. raoultii and its candidate vector tick species, which suggest an emerged potential threat of human health in the areas.     

Application of quantitative measures of gene order similarity to phylogenetic reconstructions (exemplified by bacteria of the genus <Emphasis Type="Italic">Rickettsia</Emphasis>)

Data reflecting evolutionary changes in chromosomal gene order can be used for phylogenetic reconstructions along with the results of nucleotide sequence comparison. By the example of bacteria of the genus Rickettsia, we have shown that phylogenetic reconstructions based on quantitative estimates of the similarity and cladistic analysis of gene order data, may, in some cases, amend and fill up classical phylogenetic trees. When applied, these approaches enabled us to substantiate the hypothesis that Rickettsia felis species had split before the typhus (R. typhi, R. prowazekii) and spotted fever (R. connorii) group divergence and thus R. felis does not belong to the latter group. In general, rickettsias evolved towards increasing intracellular parasitic specialization. Five Rickettsia species whose genomes have been sequenced and annotated completely actually form an evolutionary series R. bellii—R. felis—R. conorii—R. prowazekii—R. typhi. Within this series, a reduction in genome size and rapid decrease of genome rearrangement rates (genome plasticity loss) gradually occur.     

Rickettsia Phylogenomics: Unwinding the Intricacies of Obligate Intracellular Life

Background

Completed genome sequences are rapidly increasing for Rickettsia, obligate intracellular α-proteobacteria responsible for various human diseases, including epidemic typhus and Rocky Mountain spotted fever. In light of phylogeny, the establishment of orthologous groups (OGs) of open reading frames (ORFs) will distinguish the core rickettsial genes and other group specific genes (class 1 OGs or C1OGs) from those distributed indiscriminately throughout the rickettsial tree (class 2 OG or C2OGs).

Methodology/Principal Findings

We present 1823 representative (no gene duplications) and 259 non-representative (at least one gene duplication) rickettsial OGs. While the highly reductive (∼1.2 MB) Rickettsia genomes range in predicted ORFs from 872 to 1512, a core of 752 OGs was identified, depicting the essential Rickettsia genes. Unsurprisingly, this core lacks many metabolic genes, reflecting the dependence on host resources for growth and survival. Additionally, we bolster our recent reclassification of Rickettsia by identifying OGs that define the AG (ancestral group), TG (typhus group), TRG (transitional group), and SFG (spotted fever group) rickettsiae. OGs for insect-associated species, tick-associated species and species that harbor plasmids were also predicted. Through superimposition of all OGs over robust phylogeny estimation, we discern between C1OGs and C2OGs, the latter depicting genes either decaying from the conserved C1OGs or acquired laterally. Finally, scrutiny of non-representative OGs revealed high levels of split genes versus gene duplications, with both phenomena confounding gene orthology assignment. Interestingly, non-representative OGs, as well as OGs comprised of several gene families typically involved in microbial pathogenicity and/or the acquisition of virulence factors, fall predominantly within C2OG distributions.

Conclusion/Significance

Collectively, we determined the relative conservation and distribution of 14354 predicted ORFs from 10 rickettsial genomes across robust phylogeny estimation. The data, available at PATRIC (PathoSystems Resource Integration Center), provide novel information for unwinding the intricacies associated with Rickettsia pathogenesis, expanding the range of potential diagnostic, vaccine and therapeutic targets.     

Genome sequences published outside of Standards in Genomic Sciences, March-April 2012

The purpose of this table is to provide the community with a citable record of publications of ongoing genome sequencing projects that have led to a publication in the scientific literature. While our goal is to make the list complete, there is no guarantee that we may have omitted one or more publications appearing in this time frame. Readers and authors who wish to have publications added to subsequent versions of this list are invited to provide the bibliographic data for such references to the SIGS editorial office.

Phylum Euryarchaeota

• Halococcus hamelinensis, sequence accession PRJNA80845 [1]
• “Methanocella conradii” HZ254, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"CP003243","term_id":"379319823","term_text":"CP003243"}}CP003243 [2]
• Thermococcus litoralis NS-C, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AHVB00000000","term_id":"374743520","term_text":"AHVB00000000"}}AHVB00000000 [3]

Phylum Crenarchaeota

• Candidatus Nitrosopumilus salaria” BD31, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AEXL00000000","term_id":"386807666","term_text":"AEXL00000000"}}AEXL00000000 [4]
• Candidatus Nitrosoarchaeum limnia, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AHJG00000000","term_id":"367466343","term_text":"AHJG00000000"}}AHJG00000000 [5]

Phylum Deinococcus-Thermus

• Deinococcus gobiensis, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"CP002536","term_id":"324314064","term_text":"CP002536"}}CP002536 [6]

Phylum Proteobacteria

• Aggregatibacter actinomycetemcomitans strain ANH9381, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"CP003099","term_id":"365745154","term_text":"CP003099"}}CP003099 [7]
• Alishewanella jeotgali, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AHTH00000000","term_id":"374571619","term_text":"AHTH00000000"}}AHTH00000000 [8]
• Enterobacter aerogenes KCTC 2190, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"CP002824","term_id":"334732565","term_text":"CP002824"}}CP002824 [9]
• Escherichia coli O104:H4, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AFOB02000092","term_id":"340739517","term_text":"AFOB02000092"}}AFOB02000092 [10]
• Helicobacter pylori strains 17874, sequence accession PRJNA76569 [11]
• Helicobacter pylori strains P79, sequence accession PRJNA76567 [11]
• Janthinobacterium sp. Strain PAMC 25724, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AHHB00000000","term_id":"372292151","term_text":"AHHB00000000"}}AHHB00000000 [12]
• Klebsiella oxytoca KCTC 1686, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"CP003218","term_id":"365906294","term_text":"CP003218"}}CP003218 [13]
• Klebsiella pneumoniae subsp. pneumoniae HS11286, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"CP003200","term_id":"364515570","term_text":"CP003200"}}CP003200 (chromosome), {"type":"entrez-nucleotide","attrs":{"text":"CP003223","term_id":"365803686","term_text":"CP003223"}}CP003223 (plasmid pKPHS1), {"type":"entrez-nucleotide","attrs":{"text":"CP003224","term_id":"365803828","term_text":"CP003224"}}CP003224 (plasmid pKPHS2), {"type":"entrez-nucleotide","attrs":{"text":"CP003225","term_id":"365803989","term_text":"CP003225"}}CP003225 (plasmid pKPHS3), {"type":"entrez-nucleotide","attrs":{"text":"CP003226","term_id":"365804142","term_text":"CP003226"}}CP003226 (plasmid pKPHS4), {"type":"entrez-nucleotide","attrs":{"text":"CP003227","term_id":"365804147","term_text":"CP003227"}}CP003227 (plasmid pKPHS5), {"type":"entrez-nucleotide","attrs":{"text":"CP003228","term_id":"365804153","term_text":"CP003228"}}CP003228 (plasmid pKPHS6) [14]
• Oceanimonas sp. GK1, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"CP003171","term_id":"372983507","term_text":"CP003171"}}CP003171 [15]
• “Pseudogulbenkiania ferrooxidans” Strain 2002, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"NZ_ACIS01000000","term_id":"224827334","term_text":"ref||NZ_ACIS01000000"}}NZ_ACIS01000000 [16]
• Pseudomonas extremaustralis 14-3b, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AHIP00000000","term_id":"371721769","term_text":"AHIP00000000"}}AHIP00000000 [17]
• Pseudomonas sp. Strain PAMC 25886, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AHHC00000000","term_id":"372002201","term_text":"AHHC00000000"}}AHHC00000000 [18]
• Psychrobacter, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AHVZ00000000","term_id":"375126956","term_text":"AHVZ00000000"}}AHVZ00000000 [19]
• Rahnella sp. Strain Y9602, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"CP002505","term_id":"321165934","term_text":"CP002505"}}CP002505 [20]
• Rhizobium sp. Strain PDO1-076, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AHZC00000000","term_id":"375058045","term_text":"AHZC00000000"}}AHZC00000000 [21]
• Rhodospirillum photometricum DSM122, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"HE663493","term_id":"378401447","term_text":"HE663493"}}HE663493 [22]
• “Rickettsia sibirica sibirica”, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AHIZ00000000","term_id":"374669031","term_text":"AHIZ00000000"}}AHIZ00000000 [23]
• Rickettsia sibirica subsp. mongolitimonae strain HA-91, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AHZB00000000","term_id":"375318263","term_text":"AHZB00000000"}}AHZB00000000 [24]
• Salmonella enterica subsp. enterica Serotype Enteritidis Strain LA5, sequence accession [25]
• Salmonella enterica subsp. enterica Serotype Senftenberg Strain SS209, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"CAGQ00000000","term_id":"379987603","term_text":"CAGQ00000000"}}CAGQ00000000 [26]
• Salmonella enterica subsp. enterica Serovar Typhi P-stx-12, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"CP003278","term_id":"374352002","term_text":"CP003278"}}CP003278 (chromosome) and {"type":"entrez-nucleotide","attrs":{"text":"CP003279","term_id":"374356693","term_text":"CP003279"}}CP003279 (plasmid) [27]
• Sphingomonas echinoides ATCC 14820, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AHIR00000000","term_id":"366040205","term_text":"AHIR00000000"}}AHIR00000000 [28]
• Strain HIMB55, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AGIF00000000","term_id":"374304583","term_text":"AGIF00000000"}}AGIF00000000 [29]
• Vibrio harveyi CAIM 1792, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AHHQ00000000","term_id":"374671508","term_text":"AHHQ00000000"}}AHHQ00000000 [30]
• Wolbachia Strain wAlbB, sequence accession {"type":"entrez-nucleotide-range","attrs":{"text":"CAGB01000001 to CAGB01000165","start_term":"CAGB01000001","end_term":"CAGB01000165","start_term_id":"371933004","end_term_id":"371931774"}}CAGB01000001 to CAGB01000165 [31]
• Xanthomonas axonopodis pv. punicae Strain LMG 859, sequence accession {"type":"entrez-nucleotide-range","attrs":{"text":"CAGJ01000001 to CAGJ01000217","start_term":"CAGJ01000001","end_term":"CAGJ01000217","start_term_id":"372556075","end_term_id":"372551622"}}CAGJ01000001 to CAGJ01000217 [32]

Phylum Tenericutes

• Mycoplasma hyorhinis Strain GDL-1, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"CP003231","term_id":"367460291","term_text":"CP003231"}}CP003231 [33]

Phylum Firmicutes

• Bacillus subtilis, sequence accession BGSCID 3A27 through BGSCID 28A4 [34]
• Clostridium difficile Strain CD37, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AHJJ00000000","term_id":"371930118","term_text":"AHJJ00000000"}}AHJJ00000000 [35]
• Clostridium perfringens, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AFES00000000","term_id":"380306484","term_text":"AFES00000000"}}AFES00000000 [36]
• Lactobacillus fructivorans KCTC 3543, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AEQY00000000","term_id":"316986063","term_text":"AEQY00000000"}}AEQY00000000 [37]
• Lactococcus lactis IO-1, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AP012281","term_id":"374672113","term_text":"AP012281"}}AP012281 [38]
• Lactobacillus plantarum strain NC8, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AGRI00000000","term_id":"376011245","term_text":"AGRI00000000"}}AGRI00000000 [39]
• Paenibacillus dendritiformis C454, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AHKH00000000","term_id":"374393106","term_text":"AHKH00000000"}}AHKH00000000 [40]
• Paenibacillus sp. Strain Aloe-11, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AGFI00000000","term_id":"374477730","term_text":"AGFI00000000"}}AGFI00000000 [41]
• “Peptoniphilus rhinitidis” 1-13^T, sequence accession {"type":"entrez-nucleotide-range","attrs":{"text":"BAEW01000001 to BAEW01000056","start_term":"BAEW01000001","end_term":"BAEW01000056","start_term_id":"374722129","end_term_id":"374722074"}}BAEW01000001 to BAEW01000056 [42]
• Streptococcus macedonicus ACA-DC 198, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"HE613569","term_id":"372283141","term_text":"HE613569"}}HE613569 and {"type":"entrez-nucleotide","attrs":{"text":"HE613570","term_id":"372285142","term_text":"HE613570"}}HE613570 [43]
• Staphylococcus aureus VC40, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"CP003033","term_id":"374362062","term_text":"CP003033"}}CP003033 [44]
• Streptococcus infantarius subsp. infantarius Strain CJ18, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"CP003295","term_id":"374681091","term_text":"CP003295"}}CP003295 (chromosome), {"type":"entrez-nucleotide","attrs":{"text":"CP003296","term_id":"374682963","term_text":"CP003296"}}CP003296 (plasmid) [45]
• Streptococcus macedonicus ACA-DC 198, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"HE613569","term_id":"372283141","term_text":"HE613569"}}HE613569 (chromosome), {"type":"entrez-nucleotide","attrs":{"text":"HE613570","term_id":"372285142","term_text":"HE613570"}}HE613570 (plasmid pSMA198) [46]

Phylum Actinobacteria

• Actinoplanes sp. SE50/110, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"CP003170","term_id":"359832573","term_text":"CP003170"}}CP003170 [47]
• Amycolatopsis sp. Strain ATCC 39116, sequence accession [48]
• Nocardia cyriacigeorgica GUH-2, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"FO082843","term_id":"374843763","term_text":"FO082843"}}FO082843 [49]
• Salinibacterium sp., sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AHWA00000000","term_id":"375323651","term_text":"AHWA00000000"}}AHWA00000000 [50]
• Streptomyces acidiscabies 84-104, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AHBF00000000","term_id":"372137622","term_text":"AHBF00000000"}}AHBF00000000 [51]

Non-Bacterial genomes

• Bluetongue Virus Serotype 2, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AJ783905","term_id":"70907482","term_text":"AJ783905"}}AJ783905 (Seg-6) and {"type":"entrez-nucleotide","attrs":{"text":"JQ681257","term_id":"383932043","term_text":"JQ681257"}}JQ681257 (Seg-1), {"type":"entrez-nucleotide","attrs":{"text":"JQ681257","term_id":"383932043","term_text":"JQ681257"}}JQ681257 (Seg-1), {"type":"entrez-nucleotide","attrs":{"text":"JQ681258","term_id":"383932045","term_text":"JQ681258"}}JQ681258 (Seg-2), {"type":"entrez-nucleotide","attrs":{"text":"JQ681259","term_id":"383932047","term_text":"JQ681259"}}JQ681259 (Seg-3), {"type":"entrez-nucleotide","attrs":{"text":"JQ681260","term_id":"383932049","term_text":"JQ681260"}}JQ681260 (Seg-4), {"type":"entrez-nucleotide","attrs":{"text":"JQ681261","term_id":"383932051","term_text":"JQ681261"}}JQ681261 (Seg-5), {"type":"entrez-nucleotide","attrs":{"text":"JQ681262","term_id":"383932053","term_text":"JQ681262"}}JQ681262 (Seg-7), JQ6812563 (Seg-8), JQ6812564 (Seg-9), to {"type":"entrez-nucleotide","attrs":{"text":"JQ681265","term_id":"383932059","term_text":"JQ681265"}}JQ681265 (Seg-10) [52]
• Virus Serotype 1, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AJ585111","term_id":"118420282","term_text":"AJ585111"}}AJ585111 (Seg-2), {"type":"entrez-nucleotide","attrs":{"text":"AJ586659","term_id":"47826214","term_text":"AJ586659"}}AJ586659 (Seg-6), {"type":"entrez-nucleotide","attrs":{"text":"JQ282770","term_id":"383215071","term_text":"JQ282770"}}JQ282770 (Seg-1), {"type":"entrez-nucleotide","attrs":{"text":"JQ282771","term_id":"383215073","term_text":"JQ282771"}}JQ282771 (Seg-3), {"type":"entrez-nucleotide","attrs":{"text":"JQ282772","term_id":"383215075","term_text":"JQ282772"}}JQ282772 (Seg-4), {"type":"entrez-nucleotide","attrs":{"text":"JQ282773","term_id":"383215077","term_text":"JQ282773"}}JQ282773 (Seg-5), {"type":"entrez-nucleotide","attrs":{"text":"JQ282774","term_id":"383215079","term_text":"JQ282774"}}JQ282774 (Seg-7), {"type":"entrez-nucleotide","attrs":{"text":"JQ282775","term_id":"383215081","term_text":"JQ282775"}}JQ282775 (Seg-8), {"type":"entrez-nucleotide","attrs":{"text":"JQ282776","term_id":"383215083","term_text":"JQ282776"}}JQ282776 (Seg-9), and {"type":"entrez-nucleotide","attrs":{"text":"JQ282777","term_id":"383215085","term_text":"JQ282777"}}JQ282777 (Seg-10) [52]
• Chloroplast genome of Erycina pusilla, sequence accession JF_746994 [53]
• Danio rerio, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"JQ434101","term_id":"384872525","term_text":"JQ434101"}}JQ434101 [54]
• Enterococcal Bacteriophage SAP6, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"JF731128","term_id":"343411857","term_text":"JF731128"}}JF731128 [55]
• Eubenangee virus, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"JQ070376","term_id":"383513837","term_text":"JQ070376"}}JQ070376 through {"type":"entrez-nucleotide","attrs":{"text":"JQ070385","term_id":"383513855","term_text":"JQ070385"}}JQ070385 [56]
• Fujian/411-like viruses, sequence accession {"type":"entrez-nucleotide-range","attrs":{"text":"CY087969 to CY088568","start_term":"CY087969","end_term":"CY088568","start_term_id":"380506411","end_term_id":"380507803"}}CY087969 to CY088568 [57]
• Hantavirus Variant of Rio Mamoré Virus, Maripa Virus, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"JQ611712","term_id":"384382977","term_text":"JQ611712"}}JQ611712 (segment S), {"type":"entrez-nucleotide","attrs":{"text":"JQ611713","term_id":"384382979","term_text":"JQ611713"}}JQ611713 (segment M), and {"type":"entrez-nucleotide","attrs":{"text":"JQ611714","term_id":"384382981","term_text":"JQ611714"}}JQ611714 (segment L) [58]
• Pata virus, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"JQ070386","term_id":"383513857","term_text":"JQ070386"}}JQ070386 through {"type":"entrez-nucleotide","attrs":{"text":"JQ070395","term_id":"383513875","term_text":"JQ070395"}}JQ070395 [59]
• Porcine Circovirus 2, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"JQ413808","term_id":"383215164","term_text":"JQ413808"}}JQ413808 [60]
• Porcine Reproductive and Respiratory Syndrome Virus, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"JQ326271","term_id":"382929416","term_text":"JQ326271"}}JQ326271 [61]
• Streptococcus mutans Phage M102AD, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"DQ386162","term_id":"88698119","term_text":"DQ386162"}}DQ386162 [62]
• Tilligery virus, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"JQ070366","term_id":"383513817","term_text":"JQ070366"}}JQ070366 through {"type":"entrez-nucleotide","attrs":{"text":"JQ070375","term_id":"383513835","term_text":"JQ070375"}}JQ070375 [63]

     

No Evidence for Lymphatic Filariasis Transmission in Big Cities Affected by Conflict Related Rural-Urban Migration in Sierra Leone and Liberia

Background

In West Africa, the principal vectors of lymphatic filariasis (LF) are Anopheles species with Culex species playing only a minor role in transmission, if any. Being a predominantly rural disease, the question remains whether conflict-related migration of rural populations into urban areas would be sufficient for active transmission of the parasite.

Methodology/Principal Findings

We examined LF transmission in urban areas in post-conflict Sierra Leone and Liberia that experienced significant rural-urban migration. Mosquitoes from Freetown and Monrovia, were analyzed for infection with Wuchereria bancrofti. We also undertook a transmission assessment survey (TAS) in Bo and Pujehun districts in Sierra Leone. The majority of the mosquitoes collected were Culex species, while Anopheles species were present in low numbers. The mosquitoes were analyzed in pools, with a maximum of 20 mosquitoes per pool. In both countries, a total of 1731 An. gambiae and 14342 Culex were analyzed for W. bancrofti, using the PCR. Two pools of Culex mosquitoes and 1 pool of An. gambiae were found infected from one community in Freetown. Pool screening analysis indicated a maximum likelihood of infection of 0.004 (95% CI of 0.00012–0.021) and 0.015 (95% CI of 0.0018–0.052) for the An. gambiae and Culex respectively. The results indicate that An. gambiae is present in low numbers, with a microfilaria prevalence breaking threshold value not sufficient to maintain transmission. The results of the TAS in Bo and Pujehun also indicated an antigen prevalence of 0.19% and 0.67% in children, respectively. This is well below the recommended 2% level for stopping MDA in Anopheles transmission areas, according to WHO guidelines.

Conclusions

We found no evidence for active transmission of LF in cities, where internally displaced persons from rural areas lived for many years during the more than 10 years conflict in Sierra Leone and Liberia.     

Origin and Evolution of Rickettsial Plasmids

Background

Rickettsia species are strictly intracellular bacteria that have undergone a reductive genomic evolution. Despite their allopatric lifestyle, almost half of the 26 currently validated Rickettsia species have plasmids. In order to study the origin, evolutionary history and putative roles of rickettsial plasmids, we investigated the evolutionary processes that have shaped 20 plasmids belonging to 11 species, using comparative genomics and phylogenetic analysis between rickettsial, microbial and non-microbial genomes.

Results

Plasmids were differentially present among Rickettsia species. The 11 species had 1 to 4 plasmid (s) with a size ranging from 12 kb to 83 kb. We reconstructed pRICO, the last common ancestor of the current rickettsial plasmids. pRICO was vertically inherited mainly from Rickettsia/Orientia chromosomes and diverged vertically into a single or multiple plasmid(s) in each species. These plasmids also underwent a reductive evolution by progressive gene loss, similar to that observed in rickettsial chromosomes, possibly leading to cryptic plasmids or complete plasmid loss. Moreover, rickettsial plasmids exhibited ORFans, recent gene duplications and evidence of horizontal gene transfer events with rickettsial and non-rickettsial genomes mainly from the α/γ-proteobacteria lineages. Genes related to maintenance and plasticity of plasmids, and to adaptation and resistance to stress mostly evolved under vertical and/or horizontal processes. Those involved in nucleotide/carbohydrate transport and metabolism were under the influence of vertical evolution only, whereas genes involved in cell wall/membrane/envelope biogenesis, cycle control, amino acid/lipid/coenzyme and secondary metabolites biosynthesis, transport and metabolism underwent mainly horizontal transfer events.

Conclusion

Rickettsial plasmids had a complex evolution, starting with a vertical inheritance followed by a reductive evolution associated with increased complexity via horizontal gene transfer as well as gene duplication and genesis. The plasmids are plastic and mosaic structures that may play biological roles similar to or distinct from their co-residing chromosomes in an obligate intracellular lifestyle.     

Apolipophorin-III mediates antiplasmodial epithelial responses in Anopheles gambiae (G3) mosquitoes

Background

Apolipophorin-III (ApoLp-III) is known to play an important role in lipid transport and innate immunity in lepidopteran insects. However, there is no evidence of involvement of ApoLp-IIIs in the immune responses of dipteran insects such as Drosophila and mosquitoes.

Methodology/Principal Findings

We report the molecular and functional characterization of An. gambiae apolipophorin-III (AgApoLp-III). Mosquito ApoLp-IIIs have diverged extensively from those of lepidopteran insects; however, the predicted tertiary structure of AgApoLp-III is similar to that of Manduca sexta (tobacco hornworm). We found that AgApoLp-III mRNA expression is strongly induced in the midgut of An. gambiae (G3 strain) mosquitoes in response to Plasmodium berghei infection. Furthermore, immunofluorescence stainings revealed that high levels of AgApoLp-III protein accumulate in the cytoplasm of Plasmodium-invaded cells and AgApoLp-III silencing increases the intensity of P. berghei infection by five fold.

Conclusion

There are broad differences in the midgut epithelial responses to Plasmodium invasion between An. gambiae strains. In the G3 strain of An. gambiae AgApoLp-III participates in midgut epithelial defense responses that limit Plasmodium infection.     

A New Long-Lasting Indoor Residual Formulation of the Organophosphate Insecticide Pirimiphos Methyl for Prolonged Control of Pyrethroid-Resistant Mosquitoes: An Experimental Hut Trial in Benin

Background

Indoor residual spraying (IRS) is widely used for malaria transmission control in sub-Saharan Africa. Resistance to pyrethroids in the mosquito Anopheles gambiae is a growing problem. There is an urgent need to develop long-lasting alternative insecticides to reduce selection pressure for pyrethroid resistance and to provide control with a single IRS application in countries with long transmission seasons.

Methods

Two capsule suspension formulations (CS) of the organophosphate pirimiphos methyl were evaluated as IRS treatments in experimental huts in an area of Benin where the mosquitoes Anopheles gambiae and Culex quinquefasciatus are resistant to pyrethroids but susceptible to organophosphates. The CS formulations were tested alongside an emulsifiable concentrate (EC) formulation of pirimiphos methyl and a CS formulation of the pyrethroid lambdacyhalothrin.

Results

The two CS formulations of pirimiphos methyl gave prolonged control of An. gambiae and Cx. quinquefasciatus. In cement huts application rates of 0.5 g/m² induced high mortality of An. gambiae for almost a year: overall mortality rates 87% (95% CI 82–91%) and 92% (95% CI 88–94%). In mud huts application rates of 1 g/m² induced high mortality of An. gambiae for 10 months: overall mortality rates 75% (95% CI 69–81%) and 76% (95% CI 68–83%). The EC formulation of pirimiphos methyl failed to control An. gambiae two months after spraying. The pyrethroid lambdacyhalothrin demonstrated prolonged residual activity in bioassay tests but failed to control pyrethroid resistant An. gambiae that entered the huts. Pirimiphos methyl CS was highly active against Culex quinquefasciatus and gave control for 10 months in cement huts and 6 months in mud huts.

Conclusion

Pirimiphos methyl CS (Actellic 300 CS) applied at 1 g/m² shows great promise for providing prolonged control of pyrethroid-resistant An gambiae and for delaying pyrethroid resistance. An alternative to DDT, giving year-round transmission control in sub-Saharan Africa is now a realistic prospect.     

Beer Consumption Increases Human Attractiveness to Malaria Mosquitoes

Background

Malaria and alcohol consumption both represent major public health problems. Alcohol consumption is rising in developing countries and, as efforts to manage malaria are expanded, understanding the links between malaria and alcohol consumption becomes crucial. Our aim was to ascertain the effect of beer consumption on human attractiveness to malaria mosquitoes in semi field conditions in Burkina Faso.

Methodology/Principal Findings

We used a Y tube-olfactometer designed to take advantage of the whole body odour (breath and skin emanations) as a stimulus to gauge human attractiveness to Anopheles gambiae (the primary African malaria vector) before and after volunteers consumed either beer (n = 25 volunteers and a total of 2500 mosquitoes tested) or water (n = 18 volunteers and a total of 1800 mosquitoes). Water consumption had no effect on human attractiveness to An. gambiae mosquitoes, but beer consumption increased volunteer attractiveness. Body odours of volunteers who consumed beer increased mosquito activation (proportion of mosquitoes engaging in take-off and up-wind flight) and orientation (proportion of mosquitoes flying towards volunteers'' odours). The level of exhaled carbon dioxide and body temperature had no effect on human attractiveness to mosquitoes. Despite individual volunteer variation, beer consumption consistently increased attractiveness to mosquitoes.

Conclusions/Significance

These results suggest that beer consumption is a risk factor for malaria and needs to be integrated into public health policies for the design of control measures.     

Hydrolyzing Proficiency of Keratinases in Feather Degradation

The keratinase degrade highly rigid, cross linked structural polypeptides with different efficiency depending on the type of source. Two newly isolated strains of Bacillus subtilis (RSE163 and RSE165; NCBI Accession no {"type":"entrez-nucleotide","attrs":{"text":"JQ887983","term_id":"453198585","term_text":"JQ887983"}}JQ887983 and {"type":"entrez-nucleotide","attrs":{"text":"JQ887982","term_id":"453198574","term_text":"JQ887982"}}JQ887982) were found to be efficient keratinase producers with unusual catalytic activity result in different morphological changes in degradation pattern of feather, confirmed by their scanned electron micrographs. Maximum keratinolytic activity of both the strains B. subtilis RSE163 and RSE165 were found to be 366 ± 15.79 and 194 ± 7.26 U after 72 h of incubation. While the disulphide reductase activity of RSE163 and RSE165 estimated 0.24 ± 0.05 and 0.15 ± 0.03 U/ml of enzyme after 24 h of incubation. A total of 16 free amino acids of variable concentration were also analyzed in the cell free supernatant of hydrolyzed feather from two strains. Present study demonstrates the action of two different keratinases in feather degradation.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-014-0477-5) contains supplementary material, which is available to authorized users.     

Olyset Duo? (a Pyriproxyfen and Permethrin Mixture Net): An Experimental Hut Trial against Pyrethroid Resistant Anopheles gambiae and Culex quinquefasciatus in Southern Benin

Background

Alternative compounds which can complement pyrethroids on long-lasting insecticidal nets (LN) in the control of pyrethroid resistant malaria vectors are urgently needed. Pyriproxyfen (PPF), an insect growth regulator, reduces the fecundity and fertility of adult female mosquitoes. LNs containing a mixture of pyriproxyfen and pyrethroid could provide personal protection through the pyrethroid component and reduce vector abundance in the next generation through the sterilizing effect of pyriproxyfen.

Method

The efficacy of Olyset Duo, a newly developed mixture LN containing pyriproxyfen and permethrin, was evaluated in experimental huts in southern Benin against pyrethroid resistant Anopheles gambiae and Culex quinquefasciatus. Comparison was made with Olyset Net® (permethrin alone) and a LN with pyriproxyfen alone (PPF LN). Laboratory tunnel tests were performed to substantiate the findings in the experimental huts.

Results

Overall mortality of wild pyrethroid resistant An. gambiae s.s. was significantly higher with Olyset Duo than with Olyset Net (50% vs. 27%, P = 0.01). Olyset DUO was more protective than Olyset Net (71% vs. 3%, P<0.001). The oviposition rate of surviving blood-fed An. gambiae from the control hut was 37% whereas none of those from Olyset Duo and PPF LN huts laid eggs. The tunnel test results were consistent with the experimental hut results. Olyset Duo was more protective than Olyset Net in the huts against wild pyrethroid resistant Cx. quinquefasciatus although mortality rates of this species did not differ significantly between Olyset Net and Olyset Duo. There was no sterilizing effect on surviving blood-fed Cx. quinquefasciatus with the PPF-treated nets.

Conclusion

Olyset Duo was superior to Olyset Net in terms of personal protection and killing of pyrethroid resistant An. gambiae, and sterilized surviving blood-fed mosquitoes. Mixing pyrethroid and pyriproxyfen on a LN shows potential for malaria control and management of pyrethroid resistant vectors by preventing further selection of pyrethroid resistant phenotypes.     

Transposon Insertion Reveals pRM, a Plasmid of Rickettsia monacensis

Until the recent discovery of pRF in Rickettsia felis, the obligate intracellular bacteria of the genus Rickettsia (Rickettsiales: Rickettsiaceae) were thought not to possess plasmids. We describe pRM, a plasmid from Rickettsia monacensis, which was detected by pulsed-field gel electrophoresis and Southern blot analyses of DNA from two independent R. monacensis populations transformed by transposon-mediated insertion of coupled green fluorescent protein and chloramphenicol acetyltransferase marker genes into pRM. Two-dimensional electrophoresis showed that pRM was present in rickettsial cells as circular and linear isomers. The 23,486-nucleotide (31.8% G/C) pRM plasmid was cloned from the transformant populations by chloramphenicol marker rescue of restriction enzyme-digested transformant DNA fragments and PCR using primers derived from sequences of overlapping restriction fragments. The plasmid was sequenced. Based on BLAST searches of the GenBank database, pRM contained 23 predicted genes or pseudogenes and was remarkably similar to the larger pRF plasmid. Two of the 23 genes were unique to pRM and pRF among sequenced rickettsial genomes, and 4 of the genes shared by pRM and pRF were otherwise found only on chromosomes of R. felis or the ancestral group rickettsiae R. bellii and R. canadensis. We obtained pulsed-field gel electrophoresis and Southern blot evidence for a plasmid in R. amblyommii isolate WB-8-2 that contained genes conserved between pRM and pRF. The pRM plasmid may provide a basis for the development of a rickettsial transformation vector.