Risk of Infection with Leishmania spp. in the Canine Population in the Netherlands

The dog is the main reservoir of Leishmania infantum, the causative agent of visceral leishmaniasis (VL) in humans in Southern Europe. In order to identify the risk of dogs from a Leishmania non-endemic area traveling to a Leishmania -endemic area becoming infected and the risk of transmitting infection to humans in non-endemic areas an investigation was performed, in which the results of a questionnaire were combined with the results of a serologic survey.     

Importance of Proteins Controlling Initiation of DNA Replication in the Growth of the High-Pressure-Loving Bacterium Photobacterium profundum SS9

The molecular mechanism(s) by which deep-sea bacteria grow optimally under high hydrostatic pressure at low temperatures is poorly understood. To gain further insight into the mechanism(s), a previous study screened transposon mutant libraries of the deep-sea bacterium Photobacterium profundum SS9 and identified mutants which exhibited alterations in growth at high pressure relative to that of the parent strain. Two of these mutants, FL23 (PBPRA3229::mini-Tn10) and FL28 (PBPRA1039::mini-Tn10), were found to have high-pressure sensitivity and enhanced-growth phenotypes, respectively. The PBPRA3229 and PBPRA1039 genes encode proteins which are highly similar to Escherichia coli DiaA, a positive regulator, and SeqA, a negative regulator, respectively, of the initiation of DNA replication. In this study, we investigated the hypothesis that PBPRA3229 and PBPRA1039 encode DiaA and SeqA homologs, respectively. Consistent with this, we determined that the plasmid-carried PBPRA3229 and PBPRA1039 genes restored synchrony to the initiation of DNA replication in E. coli mutants lacking DiaA and SeqA, respectively. Additionally, PBPRA3229 restored the cold sensitivity phenotype of an E. coli dnaA(Cs) diaA double mutant whereas PBPRA1039 suppressed the cold sensitivity phenotype of an E. coli dnaA(Cs) single mutant. Taken together, these findings show that the genes disrupted in FL23 and FL28 encode DiaA and SeqA homologs, respectively. Consequently, our findings add support to a model whereby high pressure affects the initiation of DNA replication in P. profundum SS9 and either the presence of a positive regulator (DiaA) or the removal of a negative regulator (SeqA) promotes growth under these conditions.Despite the fact that more than 70% of the earth''s surface is covered by oceans, which have an average temperature of 3°C and exert an average hydrostatic pressure of 38 MPa (atmospheric pressure is ∼0.1 MPa), little is understood about the molecular basis of cold- and high-pressure-adapted deep-ocean life. The discovery and isolation of the pyschrotolerant facultative piezophile (high-pressure-loving organism) Photobacterium profundum SS9 (8) have made it possible to more readily address the mechanisms of piezophilic growth at cold temperatures (for a recent review, see reference 3). P. profundum SS9 is a gammaproteobacterium originally isolated from an amphipod homogenate obtained from the Sulu Sea in the Philippines at a depth of 2.5 km and a temperature of 9°C (8). Although it grows optimally at 28 MPa and 15°C, P. profundum SS9 can also grow over a wide range of pressures (0.1 to 90 MPa) and temperatures (2 to 20°C). The ability to grow at atmospheric pressure has made P. profundum SS9 more amenable to genetic manipulation than obligate piezophiles. The P. profundum SS9 genome has been sequenced and annotated (26) and consists of two chromosomes and an 80-kb plasmid. It was determined that the 80-kb plasmid is nonessential for the piezophilic growth of P. profundum SS9 (26).To gain insights into the genetic basis of high-pressure-adapted growth, transposon mutant libraries of P. profundum SS9R (a rifampin [rifampicin]-resistant derivative of SS9) were screened in liquid culture for mutants with defects in the ability to grow at high pressure (45 MPa, 15°C) (19). One of the putative high-pressure-sensitive mutants (FL23) isolated from these screens had a mini-Tn10 insertion in the gene PBPRA3229, which encodes a protein with 75% identity (85% similarity) to Escherichia coli DiaA (DnaA initiator-associating factor) (14). Although FL23 shows growth defects at 0.1 MPa (15°C) relative to the parent strain, the ratio of growth at 45 MPa to growth at 0.1 MPa and 15°C is substantially reduced compared to that of the parent strain, confirming that disruption of PBPRA3229 results in a high-pressure sensitivity growth phenotype (19).In E. coli, DiaA is necessary to ensure the timely initiation of DNA replication (14). DiaA forms a tetramer and binds to multiple molecules of DnaA, promoting (i) the binding of DnaA to the origin of replication in E. coli (known as oriC), (ii) ATP-DnaA-specific conformational changes in the oriC complex, and (iii) the unwinding of oriC DNA (17). Consequently, E. coli DiaA acts as a positive regulator of the initiation of DNA replication. In the absence of DiaA, initiation of DNA replication is delayed and in E. coli cells with two oriC copies, it only occurs from one of these, resulting in cells with three copies of their chromosome (14). In contrast, this is an extremely rare occurrence in wild-type E. coli cells. Although disruption of diaA in E. coli results in an asynchronous DNA replication phenotype, it does not appear to affect growth or morphology at atmospheric pressure at 37°C in a genetic background with a wild-type dnaA gene. However, disruption of the diaA gene suppresses the cold sensitivity phenotype of an E. coli dnaA(Cs) mutant at 30°C.Even though PBPRA3229 is highly similar to E. coli DiaA, it also shows 45% identity (65% similarity) to a phosphoheptose isomerase in E. coli known as GmhA (4). GmhA is involved in lipopolysaccharide (LPS) biosynthesis and catalyzes the isomerization of d-sedoheptulose 7-phosphate into d-glycero-d-manno-heptose 7-phosphate, which is the first step in the biosynthesis of ADP-glycero-manno-heptose, a subunit of the LPS inner core. The LPS forms the outermost leaflet of the outer membrane of gram-negative bacterial cells, and in E. coli K-12 strains, the LPS is composed of inner and outer sugar cores and lipid A (25). E. coli K-12 mutants lacking GmhA produce truncated LPS species relative to that of the parent strain due to the absence of the inner core, which can be easily visualized by gel electrophoresis followed by silver staining (4). Due to the high degree of sequence similarity between PBPRA3229 and GmhA, it is also possible that FL23 has an alteration in its LPS relative to that of the parent strain.In contrast to DiaA, SeqA is a negative regulator of the initiation of DNA replication in E. coli (20). E. coli SeqA binds to hemimethylated oriC and prevents the binding of ATP-DnaA. Disruption of seqA in E. coli also results in an asynchronous-replication phenotype. However, the effect of DiaA on the timing of DNA replication initiation appears to be SeqA independent (14). Interestingly, a putative P. profundum SS9R seqA transposon insertion mutant (PBPRA1039::Tn10) was identified as having high-pressure-enhanced growth at 45 MPa and 15°C relative to its growth at atmospheric pressure (19). Therefore, this preliminary finding suggests that the removal of a negative regulator of the initiation of DNA replication could promote the growth of P. profundum SS9R at high pressure.In this study, we investigated the hypothesis that proteins that regulate the initiation of DNA replication play a key role in the piezophilic growth of P. profundum SS9. We determined that PBPRA3229 and PBPRA1039 encode functional DiaA and SeqA homologs, respectively, and we propose a model whereby the initiation of DNA replication is sensitive to high pressure and either the production of a positive regulator (DiaA) or the removal of a negative regulator (SeqA) can promote growth under these conditions.     

A panel of microsatellite loci from two species of octopus, Pareledone turqueti (Joubin, 1905) and Pareledone charcoti (Joubin, 1905)

Eighteen dinucleotide microsatellite loci were isolated from two octopus species, Pareledone turqueti and Pareledone charcoti, which are endemic to the Southern Ocean. Genetic diversity was assessed in samples of P. charcoti and P. turqueti from Elephant Island and Shag Rocks respectively. All except one locus (which has proved to be polymorphic in other species) were variable in the focal species and amplified between six and 30 and between four and 28 alleles for P. charcoti and P. turqueti respectively; mean expected heterozygosities varied between 0.38 and 0.95 (P. charcoti) and between 0.34 and 0.97 (P. turqueti), with significant (P < 0.05) departures from Hardy–Weinberg equilibrium at seven loci; three of these loci provided significant (P < 0.05) evidence for null alleles. Two pairs of loci isolated from P. turqueti demonstrated significant (P < 0.05) linkage disequilibrium. We are presently using these genetic markers to quantify spatial genetic structure in the genus Pareledone.     

Characterization of polymorphic microsatellites for the periwinkle gastropod, Littorina littorea (Linnaeus, 1758) and their cross-amplification in four congeners

Eight polymorphic microsatellite loci are described for Littorina littorea (Linnaeus, 1758). Data on allelic variation in Irish and Celtic Sea samples are reported. The average number of alleles per locus was 11 (range 4–29), and observed and expected heterozygosities ranged from 6.9 to 84.3% and from 9.4 to 95.2%, respectively. Loci did not deviate from Hardy–Weinberg equilibrium and no linkage disequilibrium between loci pairs was detected. Microsatellites were not highly conserved in the congeners, L. fabalis, L. saxatilis, L. compressa and L. obtusata as evidenced by a low rate of cross-amplification. These microsatellites should prove useful in population genetic studies.     

Assessment of therapeutic responses to gametocytocidal drugs in Plasmodium falciparum malaria

Background

Effective mating between laboratory-reared males and wild females is paramount to the success of vector control strategies aiming to decrease disease transmission via the release of sterile or genetically modified male mosquitoes. However mosquito colonization and laboratory maintenance have the potential to negatively affect male genotypic and phenotypic quality through inbreeding and selection, which in turn can decrease male mating competitiveness in the field. To date, very little is known about the impact of those evolutionary forces on the reproductive biology of mosquito colonies and how they ultimately affect male reproductive fitness.

Methods

Here several male reproductive physiological traits likely to be affected by inbreeding and selection following colonization and laboratory rearing were examined. Sperm length, and accessory gland and testes size were compared in male progeny from field-collected females and laboratory strains of Anopheles gambiae sensu stricto colonized from one to over 25 years ago. These traits were also compared in the parental and sequentially derived, genetically modified strains produced using a two-phase genetic transformation system. Finally, genetic crosses were performed between strains in order to distinguish the effects of inbreeding and selection on reproductive traits.

Results

Sperm length was found to steadily decrease with the age of mosquito colonies but was recovered in refreshed strains and crosses between inbred strains therefore incriminating inbreeding costs. In contrast, testes size progressively increased with colony age, whilst accessory gland size quickly decreased in males from colonies of all ages. The lack of heterosis in response to crossing and strain refreshing in the latter two reproductive traits suggests selection for insectary conditions.

Conclusions

These results show that inbreeding and selection differentially affect reproductive traits in laboratory strains overtime and that heterotic ‘supermales’ could be used to rescue some male reproductive characteristics. Further experiments are needed to establish the exact relationship between sperm length, accessory gland and testes size, and male reproductive success in the laboratory and field settings.     

Variation analysis and gene annotation of eight MHC haplotypes: The MHC Haplotype Project

The human major histocompatibility complex (MHC) is contained within about 4 Mb on the short arm of chromosome 6 and is recognised as the most variable region in the human genome. The primary aim of the MHC Haplotype Project was to provide a comprehensively annotated reference sequence of a single, human leukocyte antigen-homozygous MHC haplotype and to use it as a basis against which variations could be assessed from seven other similarly homozygous cell lines, representative of the most common MHC haplotypes in the European population. Comparison of the haplotype sequences, including four haplotypes not previously analysed, resulted in the identification of >44,000 variations, both substitutions and indels (insertions and deletions), which have been submitted to the dbSNP database. The gene annotation uncovered haplotype-specific differences and confirmed the presence of more than 300 loci, including over 160 protein-coding genes. Combined analysis of the variation and annotation datasets revealed 122 gene loci with coding substitutions of which 97 were non-synonymous. The haplotype (A3-B7-DR15; PGF cell line) designated as the new MHC reference sequence, has been incorporated into the human genome assembly (NCBI35 and subsequent builds), and constitutes the largest single-haplotype sequence of the human genome to date. The extensive variation and annotation data derived from the analysis of seven further haplotypes have been made publicly available and provide a framework and resource for future association studies of all MHC-associated diseases and transplant medicine. Horton and Gibson contributed equally to this work.     

A Vertical Wall Dominated by Acesta excavata and Neopycnodonte zibrowii,Part of an Undersampled Group of Deep-Sea Habitats

We describe a novel biotope at 633 to 762 m depth on a vertical wall in the Whittard Canyon, an extensive canyon system reaching from the shelf to the deep sea on Ireland’s continental margin. We explored this wall with an ROV and compiled a photomosaic of the habitat. The assemblage contributing to the biotope was dominated by large limid bivalves, Acesta excavata (mean shell height 10.4 cm), and deep-sea oysters, Neopycnodonte zibrowii, at high densities, particularly at overhangs. Mean density of N. zibrowii increased with depth, with densities of the most closely packed areas of A. excavata also increasing with depth. Other taxa associated with the assemblage included the solitary coral Desmophyllum dianthus, cerianthid anemones, comatulid crinoids, the trochid gastropod Margarites sp., the portunid crab Bathynectes longispina and small fish of the family Bythitidae. The scleractinian coral Madrepora oculata, the pencil urchin Cidaris cidaris and a species of Epizoanthus were also common. Prominent but less abundant species included the flytrap anemone Actinoscyphia saginata, the carrier crab Paramola cuvieri, and the fishes Lepidion eques and Conger conger. Observations of the hydrography of the canyon system identified that the upper 500 m was dominated by Eastern North Atlantic Water, with Mediterranean Outflow Water beneath it. The permanent thermocline is found between 600 and 1000 m depth, i.e., in the depth range of the vertical wall and the dense assemblage of filter feeders. Beam attenuation indicated nepheloid layers present in the canyon system with the greatest amounts of suspended material at the ROV dive site between 500 and 750 m. A cross-canyon CTD transect indicated the presence of internal waves between these depths. We hypothesise that internal waves concentrate suspended sediment at high concentrations at the foot of the vertical wall, possibly explaining the large size and high density of filter-feeding molluscs.