Vibrio harveyi phages: Isolation, characterization and evaluation of their potential as phage therapeutics on Vibrio harveyi in shrimp hatcheries

Aquaculture is a global industry providing food and employment thereby contributing to the economy. For the sustenance of aquaculture, disease management is a major requirement. Among the bacterial pathogens Vibrio harveyi remains to be the major one especially in shrimp culture systems. Rapid and mass mortality of shrimp larvae due to Vibrio harveyi infection is well known, and the pathogen causes serious economic losses in grow out systems as well. It suggests that a well defined management strategy has to be built up to protect the crop from Vibrio harveyi infection in aquaculture systems. Antibiotics have been the choice for quite some times which led to residues in meat and development of multidrug resistant bacteria which invited ban on their application. In this context several alternate options have been thought off such as probiotics, immunostimulants and vaccines. Phage therapy is yet another option. Phages being natural parasites of bacteria and are abundant in aquatic environments their application to control bacterial pathogens in aquaculture has commendable potential in lieu of antibiotics. For that matter the therapeutic effect of phages has been proven in several antibiotic resistant pathogens inclusive of Vibrio harveyi.

“Spatial and Temporal Variation of Phytoplankton Community and Their Growth Limiting Factors in Cochin Backwater”

The influence of salinity on phytoplankton varies widely, because different species have different salinity preferences. Like marine and aquatic species, many phytoplankton species exhibit tolerance to certain salinity, beyond which, it can inhibit their growth. Light is the most important factor that influences phytoplankton growth. In aquatic environments (lakes, sea or estuary) the light incident on the surface is rapidly reduced exponentially with depth (Krik, 1994). In estuaries, the major factor influencing the light availability is the suspended particulate matter, which attenuates and scatters the light. The light changes with time of the day and the season, affecting the amount of light penetrating the water column. Similarly, biological factor like copepod grazing is a major factor influencing the standing crop of phytoplankton. The copepod can actively graze up to 75% of the phytoplankton biomass in a tropical estuary (Tan et. al., 2004). It is in the context that the present study investigates the salinity, light (physical factors) and copepod grazing (biological factor) phytoplankton as the factors controlling phytoplankton growth and distribution

Heavy metal and microbial loads in sewage irrigated vegetables of Kabul, Afghanistan

Little is known about the heavy metal and microbial contamination of vegetables produced in Central Asian cities. We therefore measured the concentration of cadmium (Cd), copper (Cu), lead (Pb), and zinc (Zn) and of faecal pathogens (Coliform bacteria, Salmonella sp., Shigella sp., Ascaris lubricoides, Entamoeba sp. and pinworms [Oxyuris vermicularis syn. Enterobius vermicularis]) in soil, irrigation water, and marketed vegetables of Kabul City, Afghanistan. Leaf Pb and Zn concentrations of leafy vegetables were with 1–5 and 33–160 mg kg^{-1} dry weight (DW) several-fold above respective international thresholds of 0.3 mg Pb kg^{-1} and 50 mg Zn kg^{-1}. The tissue concentration of Cu was below threshold limits in all samples except for spinach in one farm. Above-threshold loads of microbes and parasites on vegetables were found in five out of six gardens with coliforms ranging from 0.5–2 × 10^7 cells 100g^{-1} fresh weight (FW), but no Salmonella and Shigella were found. Contamination with 0.2 × 10^7 eggs 100g^{-1} FW of Ascaris was detected on produce of three farms and critical concentrations of Entamoeba in a single case, while Oxyuris vermicularis, and Enterobius vermicularis were found on produce of three and four farms, respectively. Irrigation water had Ascaris, Coliforms, Salmonella, Shigella, Entamoeba, and Oxyuris vermicularis syn. Enterobius vermicularis ranging from 0.35 × 10^7 to 2 × 10^7 cells l^{-1}. The heavy metal and microbial loads on fresh UPA vegetables are likely the result of contamination from rising traffic, residues of the past decades of war and lacking treatment of sewage which needs urgent attention.

Effect of management strategies on the performance of ruminant livestock production systems and the safety of plant and animal products in the city of Sikasso, Mali

The regional population growth in West Africa, and especially its urban centers, will bring about new and critical challenges for urban development policy, especially in terms of ensuring food security and providing employment for the growing population. (Peri-) urban livestock and vegetable production systems, which can contribute significantly to these endeavours, are limited by various constraints, amongst them limited access to expensive production factors and their (in)efficient use. To achieve sustainable production systems with low consumer health risks, that can meet the urban increased demand, this doctoral thesis determined nutrient use efficiencies in representative (peri-) urban livestock production systems in three West African cities, and investigated potential health risks for consumers ensuing from there. The field study, which was conducted during July 2007 to December 2009, undertook a comparative analysis of (peri-) urban livestock production strategies across 210 livestock keeping households (HH) in the three West African cities of Kano/Nigeria (84 HH), Bobo Dioulasso/Burkina Faso (63 HH) and Sikasso/Mali (63 HH). These livestock enterprises were belonging to the following three farm types: commercial gardening plus field crops and livestock (cGCL; 88 HH), commercial livestock plus subsistence field cropping (cLsC; 109 HH) and commercial gardening plus semi-commercial livestock (cGscL; 13 HH) which had been classified in a preceding study; they represented the diversity of (peri-) urban livestock production systems in West Africa. In the study on the efficiency of ruminant livestock production, lactating cowsand sheep herd units were differentiated based on whether feed supplements were offered to the animals at the homestead (Go: grazing only; Gsf: mainly grazing plus some supplement feeding). Inflows and outflows of nutrients were quantified in these herds during 18 months, and the effects of seasonal variations in nutrient availability on animals’ productivity and reproductive performance was determined in Sikasso. To assess the safety of animal products and vegetables, contamination sources of irrigated lettuce and milk with microbiological contaminants, and of tomato and cabbage with pesticide residues in (peri-) urban agriculture systems of Bobo Dioulasso and Sikasso were characterized at three occasions in 2009. Samples of irrigation water, organic fertilizer and ix lettuce were collected in 6 gardens, and samples of cabbage and tomato in 12 gardens; raw and curdled milk were sampled in 6 dairy herds. Information on health risks for consumers of such foodstuffs was obtained from 11 health centers in Sikasso. In (peri-) urban livestock production systems, sheep and goats dominated (P<0.001) in Kano compared to Bobo Dioulasso and Sikasso, while cattle and poultry were more frequent (P<0.001) in Bobo Dioulasso and Sikasso than in Kano. Across cities, ruminant feeding relied on grazing and homestead supplementation with fresh grasses, crop residues, cereal brans and cotton seed cake; cereal grains and brans were the major ingredients of poultry feeds. There was little association of gardens and livestock; likewise field cropping and livestock were rarely integrated. No relation existed between the education of the HH head and the adoption of improved management practices (P>0.05), but the proportion of HH heads with a long-term experience in (peri-) urban agriculture was higher in Kano and in Bobo Dioulasso than in Sikasso (P<0.001). Cattle and sheep fetched highest market prices in Kano; unit prices for goats and chicken were highest in Sikasso. Animal inflow, outflow and dairy herd growth rates were significantly higher (P<0.05) in the Gsf than in the Go cattle herds. Maize bran and cottonseed expeller were the main feeds offered to Gsf cows as dry-season supplement, while Gsf sheep received maize bran, fresh grasses and cowpea pods. The short periodic transhumance of Go dairy cows help them maintaining their live weight, whereas Gsf cows lost weight during the dry season despite supplement feeding at a rate of 1506 g dry matter per cow and day, resulting in low productivity and reproductive performance. The daily live weight gains of calves and lambs, respectively, were low and not significantly different between the Go and the Gsf system. However, the average live weight gains of lambs were significantly higher in the dry season (P<0.05) than in the rainy season because of the high pressure of gastrointestinal parasites and of Trypanosoma sp. In consequence, 47% of the sheep leaving the Go and Gsf herds died due to diseases during the study period. Thermo-tolerant coliforms and Escherichia coli contamination levels of irrigation water significantly exceeded WHO recommendations for the unrestricted irrigation of vegetables consumed raw. Microbial contamination levels of lettuce at the farm gate and the market place in Bobo Dioulasso and at the farm gate in Sikasso were higher than at the market place in Sikasso (P<0.05). Pesticide residues were detected in only one cabbage and one tomato sample and were below the maximum residue limit for consumption. Counts of thermo-tolerant coliforms and Escherichia coli were higher in curdled than in raw milk (P<0.05). From 2006 to x 2009, cases of diarrhea/vomiting and typhoid fever had increased by 11% and 48%, respectively, in Sikasso. For ensuring economically successful and ecologically viable (peri-) urban livestock husbandry and food safety of (peri-) urban foodstuffs of animal and plant origin, the dissemination and adoption of improved feeding practices, livestock healthcare and dung management are key. In addition, measures fostering the safety of animal products and vegetables including the appropriate use of wastewater in (peri-) urban agriculture, restriction to approve vegetable pesticides and the respect of their latency periods, and passing and enforcement of safety laws is required. Finally, the incorporation of environmentally sound (peri-) urban agriculture in urban planning by policy makers, public and private extension agencies and the urban farmers themselves is of utmost importance. To enable an efficient (peri-) urban livestock production in the future, research should concentrate on cost-effective feeding systems that allow meeting the animals’ requirement for production and reproduction. Thereby focus should be laid on the use of crop-residues and leguminous forages. The improvement of the milk production potential through crossbreeding of local cattle breeds with exotic breeds known for their high milk yield might be an accompanying option, but it needs careful supervision to prevent the loss of the local trypanotolerant purebreds.

Status and Potential of Smallholder Livestock Production Systems in Xishuangbanna, southern P.R. China

With Chinas rapid economic development during the last decades, the national demand for livestock products has quadrupled within the last 20 years. Most of that increase in demand has been answered by subsidized industrialized production systems, while million of smallholders, which still provide the larger share of livestock products in the country, have been neglected. Fostering those systems would help China to lower its strong urban migration streams, enhance the livelihood of poorer rural population and provide environmentally save livestock products which have a good chance to satisfy customers demand for ecological food. Despite their importance, China’s smallholder livestock keepers have not yet gained appropriate attention from governmental authorities and researchers. However, profound analysis of those systems is required so that adequate support can lead to a better resource utilization and productivity in the sector. To this aim, this pilot study analyzes smallholder livestock production systems in Xishuangbanna, located in southern China. The area is bordered by Lao and Myanmar and geographically counts as tropical region. Its climate is characterized by dry and temperate winters and hot summers with monsoon rains from May to October. While the regionis plain, at about 500 m asl above sea level in the south, outliers of the Himalaya mountains reach out into the north of Xishuangbanna, where the highest peak reaches 2400 m asl. Except of one larger city, Jinghong, Xishuangbanna mainly is covered by tropical rainforest, areas under agricultural cultivation and villages. The major income is generated through inner-Chinese tourism and agricultural production. Intensive rubber plantations are distinctive for the lowland plains while small-scaled traditional farms are scattered in the mountane regions. In order to determine the current state and possible future chances of smallholder livestock production in that region, this study analyzed the current status of the smallholder livestock sector in the Naban River National Nature Reserve (NRNNR), an area which is largely representative for the whole prefecture. It covers an area of about 50square kilometer and reaches from 470 up to 2400 m asl. About 5500 habitants of different ethnic origin are situated in 24 villages. All data have been collected between October 2007 and May 2010. Three major objectives have been addressed in the study: 1. Classifying existing pig production systems and exploring respective pathways for development 2. Quantifying the performance of pig breeding systemsto identify bottlenecks for production 3. Analyzing past and current buffalo utilization to determine the chances and opportunities of buffalo keeping in the future In order to classify the different pig production s ystems, a baseline survey (n=204, stratified cluster sampling) was carried out to gain data about livestock species, numbers, management practices, cultivated plant species and field sizes as well associo-economic characteristics. Sampling included two clusters at village level (altitude, ethnic affiliation), resulting in 13 clusters of which 13-17 farms were interviewed respectively. Categorical Principal Component Analysis (CatPCA) and a two-step clustering algorithm have been applied to identify determining farm characteristics and assort recorded households into classes of livestock production types. The variables keep_sow_yes/no, TLU_pig, TLU_buffalo, size_of_corn_fields, altitude_class, size_of_tea_plantationand size_of_rubber_fieldhave been found to be major determinants for the characterization of the recorded farms. All farms have extensive or semi-intensive livestock production, pigs and buffaloes are predominant livestock species while chicken and aquaculture are available but play subordinate roles for livelihoods. All pig raisers rely on a single local breed, which is known as Small Ear Pig (SMEP) in the region. Three major production systemshave been identified: Livestock-corn based LB; 41%), rubber based (RB; 39%) and pig based (PB;20%) systems. RB farms earn high income from rubber and fatten 1.9 ±1.80 pigs per household (HH), often using purchased pig feed at markets. PB farms own similar sized rubber plantations and raise 4.7 ±2.77 pigs per HH, with fodder mainly being cultivated and collected in theforest. LB farms grow corn, rice and tea and keep 4.6 ±3.32 pigs per HH, also fed with collected and cultivated fodder. Only 29% of all pigs were marketed (LB: 20%; RB: 42%; PB: 25%), average annual mortality was 4.0 ±4.52 pigs per farm (LB: 4.6 ±3.68; RB: 1.9 ±2.14; PB: 7.1 ±10.82). Pig feed mainly consists of banana pseudo stem, corn and rice hives and is prepared in batches about two to three times per week. Such fodder might be sufficient in energy content but lacks appropriate content of protein. Pigs therefore suffer from malnutrition, which becomes most critical in the time before harvest season around October. Farmers reported high occurrences of gastrointestinal parasites in carcasses and often pig stables were wet and filled with manure. Deficits in nutritional and hygienic management are major limits for development and should be the first issues addressed to improve productivity. SME pork was found to be known and referred by local customers in town and by richer lowland farmers. However, high prices and lacking availability of SME pork at local wet-markets were the reasons which limited purchase. If major management constraints are overcome, pig breeders (PB and LB farms) could increase the share of marketed pigs for town markets and provide fatteners to richer RB farmers. RB farmers are interested in fattening pigs for home consumption but do not show any motivation for commercial pig raising. To determine the productivity of input factors in pig production, eproductive performance, feed quality and quantity as well as weight development of pigs under current management were recorded. The data collection included a progeny history survey covering 184 sows and 437 farrows, bi-weekly weighing of 114 pigs during a 16-months time-span on 21 farms (10 LB and 11 PB) as well as the daily recording of feed quality and quantity given to a defined number of pigs on the same 21 farms. Feed samples of all recorded ingredients were analyzed for their respective nutrient content. Since no literature values on thedigestibility of banana pseudo stem – which is a major ingredient of traditional pig feed in NRNNR – were found, a cross-sectional digestibility trial with 2x4 pigs has been conducted on a station in the research area. With the aid of PRY Herd Life Model, all data have been utilized to determine thesystems’ current (Status Quo = SQ) output and the productivity of the input factor “feed” in terms of saleable life weight per kg DM feed intake and monetary value of output per kg DM feed intake.Two improvement scenarios were simulated, assuming 1) that farmers adopt a culling managementthat generates the highest output per unit input (Scenario 1; SC I) and 2) that through improved feeding, selected parameters of reproduction are improved by 30% (SC II). Daily weight gain averaged 55 ± 56 g per day between day 200 and 600. The average feed energy content of traditional feed mix was 14.92 MJ ME. Age at first farrowing averaged 14.5 ± 4.34 months, subsequent inter-farrowing interval was 11.4 ± 2.73 months. Littersize was 5.8 piglets and weaning age was 4.3 ± 0.99 months. 18% of piglets died before weaning. Simulating pig production at actualstatus, it has been show that monetary returns on inputs (ROI) is negative (1:0.67), but improved (1:1.2) when culling management was optimized so that highest output is gained per unit feed input. If in addition better feeding, controlled mating and better resale prices at fixed dates were simulated, ROI further increased to 1:2.45, 1:2.69, 1:2.7 and 1:3.15 for four respective grower groups. Those findings show the potential of pork production, if basic measures of improvement are applied. Futureexploration of the environment, including climate, market-season and culture is required before implementing the recommended measures to ensure a sustainable development of a more effective and resource conserving pork production in the future. The two studies have shown that the production of local SME pigs plays an important role in traditional farms in NRNNR but basic constraints are limiting their productivity. However, relatively easy approaches are sufficient for reaching a notable improvement. Also there is a demand for more SME pork on local markets and, if basic constraints have been overcome, pig farmers could turn into more commercial producers and provide pork to local markets. By that, environmentally safe meat can be offered to sensitive consumers while farmers increase their income and lower the risk of external shocks through a more diverse income generating strategy. Buffaloes have been found to be the second important livestock species on NRNNR farms. While they have been a core resource of mixed smallholderfarms in the past, the expansion of rubber tree plantations and agricultural mechanization are reasons for decreased swamp buffalo numbers today. The third study seeks to predict future utilization of buffaloes on different farm types in NRNNR by analyzing the dynamics of its buffalo population and land use changes over time and calculating labor which is required for keeping buffaloes in view of the traction power which can be utilized for field preparation. The use of buffaloes for field work and the recent development of the egional buffalo population were analyzed through interviews with 184 farmers in 2007/2008 and discussions with 62 buffalo keepers in 2009. While pig based farms (PB; n=37) have abandoned buffalo keeping, 11% of the rubber based farms (RB; n=71) and 100% of the livestock-corn based farms (LB; n=76) kept buffaloes in 2008. Herd size was 2.5 ±1.80 (n=84) buffaloes in early 2008 and 2.2 ±1.69 (n=62) in 2009. Field work on own land was the main reason forkeeping buffaloes (87.3%), but lending work buffaloes to neighbors (79.0%) was also important. Other purposes were transport of goods (16.1%), buffalo trade (11.3%) and meat consumption(6.4%). Buffalo care required 6.2 ±3.00 working hours daily, while annual working time of abuffalo was 294 ±216.6 hours. The area ploughed with buffaloes remained constant during the past 10 years despite an expansion of land cropped per farm. Further rapid replacement of buffaloes by tractors is expected in the near future. While the work economy is drastically improved by the use of tractors, buffaloes still can provide cheap work force and serve as buffer for economic shocks on poorer farms. Especially poor farms, which lack alternative assets that could quickly be liquidizedin times of urgent need for cash, should not abandon buffalo keeping. Livestock has been found to be a major part of small mixed farms in NRNNR. The general productivity was low in both analyzed species, buffaloes and pigs. Productivity of pigs can be improved through basic adjustments in feeding, reproductive and hygienic management, and with external support pig production could further be commercialized to provide pork and weaners to local markets and fattening farms. Buffalo production is relatively time intensive, and only will be of importance in the future to very poor farms and such farms that cultivate very small terraces on steep slopes. These should be encouraged to further keep buffaloes. With such measures, livestock production in NRNNR has good chances to stay competitive in the future.

Wastewater usage in urban and peri-urban agricultural production systems: scenarios from India

The role urban and peri-urban agriculture (UPA) plays in reducing urban poverty and ensuring environmental sustainability was recognized by the Millennium Development Goals (MGDs). India is the world’s largest democratic nation with a population of 1.2 billion. The rapid urbanization and high proportion of people below the poverty line along with higher migration to urban areas make India vulnerable to food crisis and urbanization of poverty. Ensuring jobs and food security among urban poor is a major challenge in India. The role of UPA can be well explained and understood in this context. This paper focuses on the current situation of UPA production in India with special attention to wastewater irrigation. This question is being posed about the various human health risks from wastewater irrigation which are faced by farmers and labourers, and, secondly by consumers. The possible health hazards involve microbial pathogens as well as helminth (intestinal parasites). Based on primary and secondary data, this paper attempts to confirm that UPA is one of the best options to address increasing urban food demand and can serve to complement rural supply chains and reduce ecological food prints in India. “Good practice urban and peri-urban agriculture” necessitates an integrated approach with suitable risk reduction mechanisms to improve the efficiency and safety of UPA production.

"A Sequence in Subdomain 2 of DBL1a of Plasmodium falciparum Erythrocyte Membrane Protein 1 Induces Strain Transcending Antibodies"

Immunity to severe malaria is the first level of immunity acquired to Plasmodium falciparum. Antibodies to the variant antigen PfEMP1 (P. falciparum erythrocyte membrane protein 1) present at the surface of the parasitized red blood cell (pRBC) confer protection by blocking microvascular sequestration. Here we have generated antibodies to peptide sequences of subdomain 2 of PfEMP1-DBL1a previously identified to be associated with severe or mild malaria. A set of sera generated to the amino acid sequence KLQTLTLHQVREYWWALNRKEVWKA, containing the motif ALNRKE, stained the live pRBC. 50% of parasites tested (7/14) were positive both in flow cytometry and immunofluorescence assays with live pRBCs including both laboratory strains and in vitro adapted clinical isolates. Antibodies that reacted selectively with the sequence REYWWALNRKEVWKA in a 15-mer peptide array of DBL1a-domains were also found to react with the pRBC surface. By utilizing a peptide array to map the binding properties of the elicited anti-DBL1a antibodies, the amino acids WxxNRx were found essential for antibody binding. Complementary experiments using 135 degenerate RDSM peptide sequences obtained from 93 Ugandan patient-isolates showed that antibody binding occurred when the amino acids WxLNRKE/D were present in the peptide. The data suggests that the ALNRKE sequence motif, associated with severe malaria, induces strain-transcending antibodies that react with the pRBC surface

Annotation and characterization of the Plasmodium vivax rhoptry neck protein 4 (PvRON4)

Background: The tight junction (TJ) is one of the most important structures established during merozoite invasion of host cells and a large amount of proteins stored in Toxoplasma and Plasmodium parasites’ apical organelles are involved in forming the TJ. Plasmodium falciparum and Toxoplasma gondii apical membrane antigen 1 (AMA-1) and rhoptry neck proteins (RONs) are the two main TJ components. It has been shown that RON4 plays an essential role during merozoite and sporozoite invasion to target cells. This study has focused on characterizing a novel Plasmodium vivax rhoptry protein, RON4, which is homologous to PfRON4 and PkRON4. Methods: The ron4 gene was re-annotated in the P. vivax genome using various bioinformatics tools and taking PfRON4 and PkRON4 amino acid sequences as templates. Gene synteny, as well as identity and similarity values between open reading frames (ORFs) belonging to the three species were assessed. The gene transcription of pvron4, and the expression and localization of the encoded protein were also determined in the VCG-1 strain by molecular and immunological studies. Nucleotide and amino acid sequences obtained for pvron4 in VCG-1 were compared to those from strains coming from different geographical areas. Results: PvRON4 is a 733 amino acid long protein, which is encoded by three exons, having similar transcription and translation patterns to those reported for its homologue, PfRON4. Sequencing PvRON4 from the VCG-1 strain and comparing it to P. vivax strains from different geographical locations has shown two conserved regions separated by a low complexity variable region, possibly acting as a “smokescreen”. PvRON4 contains a predicted signal sequence, a coiled-coil α-helical motif, two tandem repeats and six conserved cysteines towards the carboxyterminus and is a soluble protein lacking predicted transmembranal domains or a GPI anchor. Indirect immunofluorescence assays have shown that PvRON4 is expressed at the apical end of schizonts and co-localizes at the rhoptry neck with PvRON2.

PvRON2, a new Plasmodium vivax rhoptry neck antigen

Background: Rhoptries are specialized organelles from parasites belonging to the phylum Apicomplexa; they secrete their protein content during invasion of host target cells and are sorted into discrete subcompartments within rhoptry neck or bulb. This distribution is associated with these proteins’ role in tight junction (TJ) and parasitophorous vacuole (PV) formation, respectively. Methods: Plasmodium falciparum RON2 amino acid sequence was used as bait for screening the codifying gene for the homologous protein in the Plasmodium vivax genome. Gene synteny, as well as identity and similarity values, were determined for ron2 and its flanking genes among P. falciparum, P. vivax and other malarial parasite genomes available at PlasmoDB and Sanger Institute databases. Pvron2 gene transcription was determined by RT-PCR of cDNA obtained from the P. vivax VCG-1 strain. Protein expression and localization were assessed by Western blot and immunofluorescence using polyclonal anti-PvRON2 antibodies. Co-localization was confirmed using antibodies directed towards specific microneme and rhoptry neck proteins. Results and discussion: The first P. vivax rhoptry neck protein (named here PvRON2) has been identified in this study. PvRON2 is a 2,204 residue-long protein encoded by a single 6,615 bp exon containing a hydrophobic signal sequence towards the amino-terminus, a transmembrane domain towards the carboxy-terminus and two coiled coil a-helical motifs; these are characteristic features of several previously described vaccine candidates against malaria. This protein also contains two tandem repeats within the interspecies variable sequence possibly involved in evading a host’s immune system. PvRON2 is expressed in late schizonts and localized in rhoptry necks similar to what has been reported for PfRON2, which suggests its participation during target cell invasion. Conclusions: The identification and partial characterization of the first P. vivax rhoptry neck protein are described in the present study. This protein is homologous to PfRON2 which has previously been shown to be associated with PfAMA-1, suggesting a similar role for PvRON2.

A sequence in subdomain 2 of DBL1 alpha of plasmodium falciparum erythrocyte membrane protein 1 induces strain transcending antibodies

Immunity to severe malaria is the first level of immunity acquired to Plasmodium falciparum. Antibodies to the variant antigen PfEMP1 (P. falciparum erythrocyte membrane protein 1) present at the surface of the parasitized red blood cell (pRBC) confer protection by blocking microvascular sequestration. Here we have generated antibodies to peptide sequences of subdomain 2 of PfEMP1-DBL1 alpha previously identified to be associated with severe or mild malaria. A set of sera generated to the amino acid sequence KLQTLTLHQVREYWWALNRKEVWKA, containing the motif ALNRKE, stained the live pRBC. 50% of parasites tested (7/14) were positive both in flow cytometry and immunofluorescence assays with live pRBCs including both laboratory strains and in vitro adapted clinical isolates. Antibodies that reacted selectively with the sequence REYWWALNRKEVWKA in a 15-mer peptide array of DBL1 alpha-domains were also found to react with the pRBC surface. By utilizing a peptide array to map the binding properties of the elicited anti-DBL1 alpha antibodies, the amino acids WxxNRx were found essential for antibody binding. Complementary experiments using 135 degenerate RDSM peptide sequences obtained from 93 Ugandan patient-isolates showed that antibody binding occurred when the amino acids WxLNRKE/D were present in the peptide. The data suggests that the ALNRKE sequence motif, associated with severe malaria, induces strain-transcending antibodies that react with the pRBC surface.

Identification, characterization and antigenicity of the Plasmodium vivax rhoptry neck protein 1 (PvRON1)

Background: Plasmodium vivax malaria remains a major health problem in tropical and sub-tropical regions worldwide. Several rhoptry proteins which are important for interaction with and/or invasion of red blood cells, such as PfRONs, Pf92, Pf38, Pf12 and Pf34, have been described during the last few years and are being considered as potential anti-malarial vaccine candidates. This study describes the identification and characterization of the P. vivax rhoptry neck protein 1 (PvRON1) and examine its antigenicity in natural P. vivax infections. Methods: The PvRON1 encoding gene, which is homologous to that encoding the P. falciparum apical sushi protein (ASP) according to the plasmoDB database, was selected as our study target. The pvron1 gene transcription was evaluated by RT-PCR using RNA obtained from the P. vivax VCG-1 strain. Two peptides derived from the deduced P. vivax Sal-I PvRON1 sequence were synthesized and inoculated in rabbits for obtaining anti-PvRON1 antibodies which were used to confirm the protein expression in VCG-1 strain schizonts along with its association with detergent-resistant microdomains (DRMs) by Western blot, and its localization by immunofluorescence assays. The antigenicity of the PvRON1 protein was assessed using human sera from individuals previously exposed to P. vivax malaria by ELISA. Results: In the P. vivax VCG-1 strain, RON1 is a 764 amino acid-long protein. In silico analysis has revealed that PvRON1 shares essential characteristics with different antigens involved in invasion, such as the presence of a secretory signal, a GPI-anchor sequence and a putative sushi domain. The PvRON1 protein is expressed in parasite's schizont stage, localized in rhoptry necks and it is associated with DRMs. Recombinant protein recognition by human sera indicates that this antigen can trigger an immune response during a natural infection with P. vivax. Conclusions: This study shows the identification and characterization of the P. vivax rhoptry neck protein 1 in the VCG-1 strain. Taking into account that PvRON1 shares several important characteristics with other Plasmodium antigens that play a functional role during RBC invasion and, as shown here, it is antigenic, it could be considered as a good vaccine candidate. Further studies aimed at assessing its immunogenicity and protection-inducing ability in the Aotus monkey model are thus recommended.

Using the PfEMP1 Head Structure Binding Motif to Deal a Blow at Severe Malaria

Plasmodium falciparum (Pf) malaria causes 200 million cases worldwide, 8 million being severe and complicated leading to similar to 1 million deaths and similar to 100,000 abortions annually. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) has been implicated in cytoadherence and infected erythrocyte rosette formation, associated with cerebral malaria; chondroitin sulphate-A attachment and infected erythrocyte sequestration related to pregnancy-associated malaria and other severe forms of disease. An endothelial cell high activity binding peptide is described in several of this similar to 300 kDa hypervariable protein's domains displaying a conserved motif (GACxPxRRxxLC); it established H-bonds with other binding peptides to mediate red blood cell group A and chondroitin sulphate attachment. This motif (when properly modified) induced PfEMP1-specific strain-transcending, fully-protective immunity for the first time in experimental challenge in Aotus monkeys, opening the way forward for a long sought-after vaccine against severe malaria.

Identification of plasmodium vivax proteins with potential role in invasion using sequence redundancy reduction and profile hidden markov models

Background: This study describes a bioinformatics approach designed to identify Plasmodium vivax proteins potentially involved in reticulocyte invasion. Specifically, different protein training sets were built and tuned based on different biological parameters, such as experimental evidence of secretion and/or involvement in invasion-related processes. A profile-based sequence method supported by hidden Markov models (HMMs) was then used to build classifiers to search for biologically-related proteins. The transcriptional profile of the P. vivax intra-erythrocyte developmental cycle was then screened using these classifiers. Results: A bioinformatics methodology for identifying potentially secreted P. vivax proteins was designed using sequence redundancy reduction and probabilistic profiles. This methodology led to identifying a set of 45 proteins that are potentially secreted during the P. vivax intra-erythrocyte development cycle and could be involved in cell invasion. Thirteen of the 45 proteins have already been described as vaccine candidates; there is experimental evidence of protein expression for 7 of the 32 remaining ones, while no previous studies of expression, function or immunology have been carried out for the additional 25. Conclusions: The results support the idea that probabilistic techniques like profile HMMs improve similarity searches. Also, different adjustments such as sequence redundancy reduction using Pisces or Cd-Hit allowed data clustering based on rational reproducible measurements. This kind of approach for selecting proteins with specific functions is highly important for supporting large-scale analyses that could aid in the identification of genes encoding potential new target antigens for vaccine development and drug design. The present study has led to targeting 32 proteins for further testing regarding their ability to induce protective immune responses against P. vivax malaria.

Análisis de la susceptibilidad de una línea celular de Aedes aegypti (Diptera: Culicidae) a la infección con Leishmania (L) chagasi y Leishmania (V) braziliensis

Se evaluó la susceptibilidad de los cultivos celulares derivados de tejidos embrionarios de Aedes aegypti a la infección con Leishmania (L) chagasi y Leishmania (V) braziliensis, agentes etiológicos de leishmaniasis visceral americana y leishmaniasis cutánea, respectivamente. Metodología: Se seleccionaron células de A. aegypti mantenidas en una mezcla de medio de cultivo Grace/L15, suplementado con suero fetal bovino al 15%, albendazol 5,4 mg/ml y una mezcla de antibióticos, e incubadas a una temperatura promedio de 26 °C. Los cultivos celulares fueron inoculados con promastigotes metacíclicos de la cepa MH/CO/84/CI-044B de L. chagasi y la cepa HOM/BR752903 de L. braziliensis en una concentración de 10 parásitos por célula. Como control positivo de la infección se utilizó la línea celular J774. Resultados: Los registros más altos en el porcentaje de infección y en el número de amastigotes por células en los cultivos celulares A. aegypti y en la línea celular J774 se obtuvieron en los días 6 y 9 pos-infección. Los resultados mostraron interacción, internalización y maduración in vitro de las dos especies del parásito en las células de este insecto no vector de Leishmania. Las células de A. aegypti infectadas mostraron cambios en el área por la influencia de los parásitos, contrario a lo registrado en las células no infectadas (P<0,05). Conclusión: Los cultivos celulares de A. aegypti emergen como un nuevo modelo in vitro para el estudio del ciclo biológico de L. chagasi y L. braziliensis.

Parasitisme i condició del lluç (Merluccius merluccius) del Golf de Lleó

El lluç europeu del Golf de Lleó és una espècie amb un gran valor comercial a les llotges catalanes per la qual cosa es troba sobreexplotat. Els paràmetres indicadors de la condició física avaluen la salut dels peixos i poden ajudar a predir la productivitat de les poblacions explotades. Un factor que pot estar relacionat amb la condició és el parasitisme, tot i que les investigacions en aquest sentit són molt escasses. Aquest estudi avalua per primera vegada la relació entre parasitisme i condició del lluç europeu. S’han examinat 30 individus, entre gener i març de 2010, i s’han localitzat paràsits que s’han classificat com a nematodes, cestodes, copèpodes i trematodes. S’han calculat també, els índexs de condició Le Cren, índex hepatosomàtic, índex gonodosomàtic i contingut lipídic en fetge i gònades. Els resultats d’aquest estudi suggereixen que els lluços infectats amb copèpodes tenen una quantitat inferior de lípids al fetge, la qual cosa podria indicar una menor condició física. Malgrat tot, cal disposar de més mostres per poder afirmar aquest fet, així com una sèrie temporal de dades que comprengui totes les estacions de l’ any