Molecular surveillance detects high prevalence of the neglected parasite Mansonella ozzardi in the Colombian Amazon.

Mansonellosis is an undermapped insect-transmitted disease caused by filarial nematodes that are estimated to infect hundreds of millions of people globally. Despite their prevalence, there are many outstanding questions regarding the general biology and health impacts of the responsible parasites. Historical reports suggest that the Colombian Amazon is endemic for mansonellosis and may serve as an ideal location to pursue these questions in the backdrop of other endemic and emerging pathogens. We deployed molecular and classical diagnostic approaches to survey Mansonella prevalence among adults belonging to indigenous communities along the Amazon River and its tributaries near Leticia, Colombia. Deployment of a loop-mediated isothermal amplification (LAMP) assay on blood samples revealed an infection prevalence of ∼40% for Mansonella ozzardi . This assay identified significantly more infections than blood smear microscopy or LAMP assays performed using plasma, likely reflecting greater sensitivity and the ability to detect low microfilaremias or occult infections. Mansonella infection rates increased with age and were higher among males compared to females. Genomic analysis confirmed the presence of M. ozzardi that clusters closely with strains sequenced in neighboring countries. We successfully cryopreserved and revitalized M. ozzardi microfilariae, advancing the prospects of rearing infective larvae in controlled settings. These data suggest an underestimation of true mansonellosis prevalence, and we expect that these methods will help facilitate the study of mansonellosis in endemic and laboratory settings.

Surgical repair of parastomal bulging: a retrospective register-based study on prospectively collected data.

AIM: The aim of this work was to examine (1) the incidence of primary repair, (2) the incidence of recurrent repair and (3) the types of repair performed in patients with parastomal bulging. METHOD: Prospectively collected data on parastomal bulging from the Danish Stoma Database were linked to surgical data on repair of parastomal bulging from the Danish National Patient Register. Survival statistics provided cumulative incidences and time until primary and recurrent repair. RESULTS: In the study sample of 1016 patients with a permanent stoma and a parastomal bulge, 180 (18%) underwent surgical repair. The cumulative incidence of a primary repair was 9% [95% CI (8%; 11%)] within 1 year and 19% [95% CI (17%; 22%)] within 5 years after the occurrence of a parastomal bulge. We found a similar probability of undergoing primary repair in patients with ileostomy and colostomy. For recurrent repair, the 5-year cumulative incidence was 5% [95% CI (3%; 7%)]. In patients undergoing repair, the probability was 33% [95% CI (21%; 46%)] of having a recurrence requiring repair within 5 years. The main primary repair was open or laparoscopic repair with mesh (43%) followed by stoma revision (39%). Stoma revision and repair with mesh could precede or follow one another as primary and recurrent repair. Stoma reversal was performed in 17% of patients. CONCLUSION: Five years after the occurrence of a parastomal bulge the estimated probability of undergoing a repair was 19%. Having undergone a primary repair, the probability of recurrent repair was high. Stoma reversal was more common than expected.

Hemocyte classification of three mosquito vectors: Aedes togoi, Anopheles lesteri and Culex quinquefasciatus.

Insect blood cells or hemocytes play an important role in the defense against parasites and other pathogenic organisms. However, the hemocyte types of three mosquito vectors, Aedes togoi, Anopheles lesteri and Culex quinquefasiatus are not well known. Therefore, the aim of this study was to characterize the hemocytes of these three mosquito species based on morphology using light microscopy. The abdominal cutting and perfusion method was used in this study as it took the fewest steps, provided the largest number of hemocytes and yielded less contamination with fat body cells. Hemocyte typing, based on morphology, revealed three types of hemocytes (prohemocytes, oenocytoids and granulocytes) that were contained in the hemolymph of all three mosquito species. This study demonstrated that the use of distinct morphology with light microscopy provided sufficient criteria to characterize and differentiate mosquito hemocytes. This technique will be useful in terms of cost saving and for new researchers who begin to study in this field.

Hemocyte classification of three mosquito vectors: Aedes togoi, Anopheles lesteri and Culex quinquefasciatus

@#Insect blood cells or hemocytes play an important role in the defense against parasites and other pathogenic organisms. However, the hemocyte types of three mosquito vectors, Aedes togoi, Anopheles lesteri and Culex quinquefasiatus are not well known. Therefore, the aim of this study was to characterize the hemocytes of these three mosquito species based on morphology using light microscopy. The abdominal cutting and perfusion method was used in this study as it took the fewest steps, provided the largest number of hemocytes and yielded less contamination with fat body cells. Hemocyte typing, based on morphology, revealed three types of hemocytes (prohemocytes, oenocytoids and granulocytes) that were contained in the hemolymph of all three mosquito species. This study demonstrated that the use of distinct morphology with light microscopy provided sufficient criteria to characterize and differentiate mosquito hemocytes. This technique will be useful in terms of cost saving and for new researchers who begin to study in this field.

The dual roles of Armigeres subalbatus prophenoloxidase V in parasite melanization and egg chorion melanization in the mosquito Ar. subalbatus.

Phenoloxidases (POs) play key roles in various physiological functions in insects, e.g., cuticular sclerotization, wound healing, egg tanning, cuticle formation and melanotic encapsulaction of pathogens. Previously, we identified five POs, designated As-pro-PO I-V, from the mosquito Armigeres subalbatus and demonstrated that the functions of As-pro-PO I, II and III, were associated with filarial parasite melanization, blood feeding and cuticle formation, respectively. In the present study, we delineate the dual functions of As-pro-PO V. We found that the level of As-pro-PO V mRNA in mosquitoes was significantly increased after microfilaria challenge or blood feeding, and decreased to normal level after oviposition. Knockdown of As-pro-PO V by dsRNA resulted in significant decreases in the degree of microfilaria melanization, egg chronic melanization rates and egg hatching rates in Ar. subalbatus. Further transfection and electrophoretic mobility-shift assays verified the As-pro-PO V gene might regulated by both AP-1, a putative immune-related regulatory element and CdxA, a developmental regulatory element. The binding of AP-1 and CdxA motif with mosquito nuclear extracts was significantly enhanced after microfilaria challenge and blood-feeding in Ar. subalbatus, respectively. These results indicate that As-pro-PO V is a critical enzyme that is required for both an effective melanization immune response and egg chorion melanization in this mosquito.

Establishment of a regional Danish database for patients with a stoma.

AIM: To present the Danish Stoma Database Capital Region with clinical variables related to stoma creation including colostomy, ileostomy and urostomy. METHOD: The stomatherapists in the Capital Region of Denmark developed a database covering patient identifiers, interventions, conditions, short-term outcome, long-term outcome and known major confounders. The completeness of data was validated against the Danish National Patient Register. RESULTS: In 2013, five hospitals included data from 1123 patients who were registered during the year. The types of stomas formed from 2007 to 2013 showed a variation reflecting the subspecialization and surgical techniques in the centres. Between 92 and 94% of patients agreed to participate in the standard programme aimed at handling of the stoma and more than 88% of patients having planned surgery had the stoma site marked pre-operatively. CONCLUSION: The database is fully operational with high data completeness and with data about patients with a stoma from before surgery up to 12 months after surgery. The database provides a solid basis for professional learning, clinical research and benchmarking.

Evaluation of a fast-track programme for patients undergoing liver resection.

BACKGROUND: Recent developments in perioperative pathophysiology and care have documented evidence-based, multimodal rehabilitation (fast-track) to hasten recovery and to decrease morbidity and hospital stay for several major surgical procedures. The aim of this study was to investigate the effect of introducing fast-track principles for perioperative care in unselected patients undergoing open or laparoscopic liver resection. METHODS: This was a prospective study involving the first 100 consecutive patients who followed fast-track principles for liver resection. Catheters and drains were systematically removed early, and patients were mobilized and started eating and drinking from the day of surgery. An opioid-sparing multimodal pain treatment was given for the first week. Discharge criteria were: pain sufficiently controlled by oral analgesics alone, patient comfortable with discharge and no untreated complications. RESULTS: Median length of stay (LOS) for all patients was 5 days, with 2 days after laparoscopic versus 5 days following open resection (P < 0·001). Median LOS after minor open resections (fewer than 3 segments) was 5 days versus 6 days for major resections (3 or more segments) (P < 0·001). Simple right or left hemihepatectomies had a median LOS of 5 days. The readmission rate was 6·0 per cent and 30-day mortality was zero. CONCLUSION: Fast-track principles for perioperative care were introduced successfully and are safe after liver resection. Routine discharge 2 days after laparoscopic resection and 4-5 days after open liver resection may be feasible.

Armigeres subalbatus (Diptera: Culicidae) prophenoloxidase III is required for mosquito cuticle formation: ultrastructural study on dsRNA-knockdown mosquitoes.

We previously suggested that Armigeres subalbatus (Coquillett) prophenoloxidase III (As-pro-PO III) might be associated with morphogenesis of larvae and pupae. Because PO and its activation system are present in the insect cuticle, and cuticle formation is a major event during pupal morphogenesis, we used ultrastructural analysis to examine the effects of As-pro-PO III knockdown on the formation of pupal and adult cuticle. Inoculation of As-pro-PO III dsRNA resulted in the incomplete formation of nascent pupal endocuticle and pharate adult cuticle, i.e., significantly fewer cuticular lamellae were deposited, the helicoidal pattern of chitin microfibrils was disorganized, and numerous electron-lucent spaces were present in the cuticular protein matrix. Similar disruptions were observed in the cuticle of adults derived from As-pro-PO III dsRNA-inoculated pupae. It has long been suggested that the quinines, generated by PO-catalyzed oxidation reactions, function as cross-linking agents; therefore, it seems reasonable to suggest that the loss of As-pro-PO III-mediated protein-protein linkages causes morphological abnormalities in the protein matrix. Our findings suggest that As-pro-PO III plays a role in cuticle formation in mosquitoes, a novel function for phenol-oxidizing enzymes.

Armigeres subalbatus prophenoloxidase III: Cloning, characterization and potential role in morphogenesis.

It has long been suggested that phenoloxidases (POs) play key roles in various physiological functions in insects, e.g., cuticular sclerotization, wound healing, egg tanning and melanotic encapsulation of pathogens. Here we report that a mosquito PO, designated Armigeres subalbatus prophenoloxidase III (As-pro-PO III), is likely involved in the morphogenesis in mosquito. Expression profile analysis found that As-pro-PO III mRNA is persistently expressed in adult mosquitoes and is not significantly affected by blood feeding, microfilariae inoculation, or Escherichia coli inoculation, but expression levels of As-pro-PO III fluctuated in larval and pupal stages. Knockdown of As-pro-PO III expression in pupae using double-stranded RNA resulted in high pupal mortality and deformed adults that subsequently died following emergence. Promoter activity analyses by electrophoretic mobility-shift assays and transfection assays suggest that the As-pro-PO III gene is positively regulated by a putative Zeste motif, a developmental regulatory element. These results suggest that As-pro-PO III is associated with morphogenesis of mosquitoes.

Profiling infection responses in the haemocytes of the mosquito, Aedes aegypti.

Pathogens that infect and/or are transmitted by mosquitoes typically are exposed to the body cavity, and to haemocytes circulating therein, during development or dissemination. Aedes aegypti haemocytes produce a range of immune response-related gene products, and an endpoint response of phagocytosis and/or melanization that is temporally and structurally distinct for the invading pathogen. Expressed sequence tags were generated from haemocyte libraries and then used to design oligonucleotide microarrays. Arrays were screened with haemocyte material collected 1-, 8- and 24-h post-inoculation with Escherichia coli or Micrococcus luteus bacteria. Data from these studies support the discovery of novel immune response-activated genes, provide an expanded understanding of antimicrobial peptide biology and highlight the coordination of immune factors that leads to an endpoint response.

Aedes aegypti phosphohexomutases and uridine diphosphate-hexose pyrophosphorylases: comparison of primary sequences, substrate specificities and temporal transcription.

Phosphohexomutases reversibly catalyse the transfer of the phosphate group of a glycosyl phosphate between the C6 and C1 positions, and uridine diphosphate (UDP)-hexose pyrophosphorylases catalyse the synthesis of UDP-hexose from uridine triphosphate (UTP) and hexose-1-phosphate. Both enzyme families are essential for nucleoside diphosphate hexose biosynthesis and are therefore critical for various physiological functions in the midgut of mosquitoes after a blood meal. We cloned and sequenced three phosphohexomutase and two UDP-hexose pyrophosphorylase cDNAs from Aedes aegypti. The products of the cDNAs were expressed and substrate specificities were examined. Herein we describe Ae. aegypti phosphoglucomutase 1, phosphoglucomutase 2, phosphoacetylglucosamine mutase, UDP-glucose pyrophosphorylase, and UDP-N-acetylglucosamine pyrophosphorylase. Transcripts of the genes expressing the enzymes are constitutively present in all life stages and blood-feeding does not seem to influence transcript abundance.

Role of dopachrome conversion enzyme in the melanization of filarial worms in mosquitoes.

Melanization is an effective defence reaction of mosquito hosts against invading parasites. In mosquitoes, the biosynthesis of melanin is initiated by the hydroxylation of tyrosine to DOPA by phenoloxidase (PO). DOPA is a branch point of the melanization reaction; it may be oxidized to dopaquinone by PO or be decarboxylated to dopamine by dopa decarboxylase. Further oxidation of dopaquinone by PO produces dopachrome. Dopachrome is then converted to 5, 6-dihydroxyindole by dopachrome conversion enzyme (DCE) to produce melanin. The conversion of dopachrome is a rate-limiting step of the melanization reaction, and the presence of PO and DCE significantly accelerates melanization reactions. In this study, a cDNA encoding DCE was cloned from the mosquito Armigeres subalbatus. Real-time PCR analysis revealed increased transcripts from haemocytes in microfilariae (mf)-inoculated mosquitoes. Gene silencing using double-stranded RNA was used to elucidate the role of DCE in the melanization reaction of parasites in Ar. subalbatus. The levels of both DCE transcripts and protein in gene knockdown mosquitoes were dramatically reduced. Compared with controls, the degree of melanization of mf in DCE-knockdown mosquitoes was significantly decreased. These results suggest that DCE is a critical enzyme that is required for effective melanization immune responses.

The use of gene silencing to study the role of dopa decarboxylase in mosquito melanization reactions.

Mosquito melanization involves hydroxylation of tyrosine to dopa, which then is oxidized to dopaquinone by phenoloxidase, or decarboxylated to dopamine by dopa decarboxlase (DDC). An Armigeres subalbatus cDNA encoding DDC was cloned and real-time PCR analysis revealed increased transcripts in blood-fed and microfilariae (mf)-inoculated mosquitoes. A double subgenomic Sindbis virus was used to silence DDC and assess its role in melanization of mf. DDC transcription and activity were significantly decreased in silenced mosquitoes, as was the degree of mf melanization 48 h postinoculation; however, melanization increased after 72 and 96 h, demonstrating that DDC influences the rate of melanization. DDC-silenced mosquitoes also exhibit high mortality, over-feeding and abnormal movement, consistent with an involvement of DDC in neurotransmission.

Melanogenesis and associated cytotoxic reactions: applications to insect innate immunity.

Insects transmit the causative agents for such debilitating diseases as malaria, lymphatic filariases, sleeping sickness, Chagas' disease, leishmaniasis, river blindness, Dengue, and yellow fever. The persistence of these diseases provides testimony to the genetic capacity of parasites to evolve strategies that ensure their successful development in two genetically diverse host species: insects and mammals. Current efforts to address the problems posed by insect-borne diseases benefit from a growing understanding of insect and mammalian immunity. Of considerable interest are recent genomic investigations that show several similarities in the innate immune effector responses and associated regulatory mechanisms manifested by insects and mammals. One notable exception, however, is the nearly universal presence of a brown-black pigment accompanying cellular innate immunity in insects. This response, which is unique to arthropods and certain other invertebrates, has focused attention on the elements involved in pigment synthesis as causing or contributing to the death of the parasite, and has even prompted speculation that the enzyme cascade mediating melanogenesis constitutes an ill-defined recognition mechanism. Experimental evidence defining the role of melanin and its precursors in insect innate immunity is severely lacking. A great deal of what is known about melanogenesis comes from studies of the process occurring in mammalian systems, where the pigment is synthesized by such diverse cells as those comprising portions of the skin, hair, inner ear, brain, and retinal epithelium. Fortunately, many of the components in the metabolic pathways leading to the formation of melanin have been found to be common to both insects and mammals. This review examines some of the factors that influence enzyme-mediated melanogenic responses, and how these responses likely contribute to blood cell-mediated, target-specific cytotoxicity in immune challenged insects.

Identification and molecular characterization of a prophenoloxidase involved in Aedes aegypti chorion melanization.

Chorion melanization is a vital biochemical event for the survival of mosquito eggs in the environment. This study describes the identification and molecular characterization of a prophenoloxidase (proPO) involved in chorion melanization in Aedes aegypti by various biochemical and molecular techniques. Results revealed that transcription of the chorion proPO occurs only in adults, blood feeding greatly stimulated its transcription and haemocytes are responsible for its transcription. Our study provides a solid basis for suggesting an essential role of the isolated proPO in chorion melanization during chorion hardening and also raises fundamental questions regarding its transportation and distribution in the chorion. This study should serve as a useful reference towards functional elucidation of other proPOs in mosquitoes.

The use of a double subgenomic Sindbis virus expression system to study mosquito gene function: effects of antisense nucleotide number and duration of viral infection on gene silencing efficiency.

Recently we established a simple, effective antisense strategy using a double subgenomic Sindbis (dsSIN) virus expression system to study gene function in mosquitoes. In this study, we further elucidate the effects of antisense nucleotide number and duration of viral infection on mosquito gene silencing efficiency by the dsSIN virus expression system. Over 15 days post virus infection, the degree of parasite melanization was progressively reduced by more than 95%, 75% and 55% in the mosquito Armigeres subalbatus transduced with 600, 147 or 36 bases antisense RNA, targeted to the highly conserved copper binding region of the Ar. subalbatus prophenoloxidase I gene (As-pro-POI), respectively. As the duration of viral infection increased from day 3-15, the degree of parasite melanization progressively decreased in all mosquitoes transduced with antisense RNA, irrespective of the lengths of antisense RNA. Progressive loss of parasite melanization function was found to correlate with down regulation of As-pro-PO expression at both the mRNA and protein activity levels, and reductions in virus titres in mosquitoes transduced with antisense RNA. A small pro-PO RNA (c. twenty-five nucleotides) was identified in mosquitoes transduced with antisense RNA. These data suggest that As-pro-POI gene expression is knocked down by degrading the As-pro-POI mRNA through the RNAi pathway. In conclusion, our study demonstrates that even a short antisense RNA (thirty-six bases) can cause silencing of the As-pro-POI gene, and the effects of endogenous gene silencing by dsSIN expression system on mosquito gene functions can be accumulative.

A novel lectin with a fibrinogen-like domain and its potential involvement in the innate immune response of Armigeres subalbatus against bacteria.

Mosquitoes have an efficient cellular innate immune response that includes phagocytosis of microbial pathogens and encapsulation of metozoan parasites. In this study, we describe a novel lectin in the mosquito, Armigeres subalbatus (aslectin or AL-1). The 1.27 kb cDNA clone for the AL-1 gene (AL-1) encodes a 279 deduced amino acid sequence that contains a C-terminal fibrinogen-like domain. AL-1 is transcribed in all life stages. AL-1 mainly exists in the haemolymph of adult female mosquitoes, and is upregulated following both Escherichia coli and Micrococcus luteus challenge. AL-1 specifically recognizes N-acetyl-d-glucosamine and is able to bind both E. coli and M. luteus. These results suggest that AL-1 might function as a pattern recognition receptor in the immune response in Ar. subalbatus.

Reassessing the role of defensin in the innate immune response of the mosquito, Aedes aegypti.

Defensin is the predominant inducible immune peptide in Aedes aegypti. In spite of its activity against Gram-positive bacteria in vitro, defensin expression is detected in mosquitoes inoculated with Gram-positive or negative bacteria, or with filarial worms. Defensin transcription and expression are dependent upon bacterial dose; however, translation is inconsistent with transcription because peptide is detectable only in mosquitoes inoculated with large doses. In vitro translation assays provide further evidence for post-transcriptional regulation of defensin. Clearance assays show that a majority of bacteria are cleared before defensin is detected. In gene silencing experiments, no significant difference in mortality was observed between defensin-deficient and control mosquitoes after bacteria inoculation. These studies suggest that defensin may have an alternative function in mosquito immunity.

A potential role for phenylalanine hydroxylase in mosquito immune responses.

In mosquitoes the melanotic encapsulation immune response is an important resistance mechanism against filarial worms and malaria parasites. The rate limiting substrate for melanin production is tyrosine that is hydroxylated by phenoloxidase (PO) to produce 3, 4-dihydroxyphenylalanine. The single pathway for endogenous production of tyrosine is by hydroxylation of phenylalanine by phenylalanine hydroxylase (PAH). In this study we describe a potential role for PAH in melanotic immune responses in the yellow fever mosquito, Aedes aegypti. A 1.6 kb A. aegypti PAH cDNA, encoding a 51 kDa protein, was isolated and subsequently expressed in an Escherichia coli expression system. In developing mosquitoes, PAH transcript is present in all stages and it is differentially expressed in adult tissues. Following an immune-challenge with Dirofilaria immitis microfilariae (mf) or bacteria, PAH transcript is up-regulated in hemocytes. Likewise, western analysis of hemocytes collected from immune-activated mosquitoes show an increase in gene product over control samples. Like PO, ultrastructure observations provide verification that PAH is located in oenocytoid and granulocyte hemocytes. Our results offer the first data that suggest PAH is used in mosquito melanin synthesis and defense responses.

Three regulatory regions of the Aedes aegypti glutamine synthetase gene differentially regulate expression: identification of a crucial regulator in the first exon.

Aedes aegyptiglutamine synthetase (GS) is expressed constitutively at various developmental stages and its relative mRNA abundance increases in the midgut following blood feeding in support of the biosynthesis of chitin, a component of the peritrophic matrix. To understand the regulation of GS expression better, GS-luciferase reporter fusion genes were constructed and analysed in transiently transfected C6/36 cells. These studies have identified three GS regions: GS-A, -B and -C (C1, C2) that are required for efficient transcription. The crucial regulatory DNA sequence is located within 140 nucleotides of the GS-C region in the first exon. GS-B region between -209 and +4 contains a negative modulator that represses transcription of the GS-C promoter, but the 5'-GS-A region, between -476 and -282, can negate the transcription inhibition of GS-B and promote GS transcription of the GS-C promoter. Electrophoretic mobility shift assays showed that nuclear proteins for GS-A, GS-B and GS-C1 are present in the C6/36 cells, and therefore that GS-A, GS-B and GS-C1 indeed possess regulatory function. By contrast, nuclear proteins isolated from both cultured cells and midgut tissues bound to GS-C2, suggesting that GS-C2 plays an important role in GS transcription and that GS-C2 is regulated by several different and redundant transcription factors to achieve constitutive expression in a wide variety of tissues.