Plasmodium falciparum and Plasmodium vivax Adjust Investment in Transmission in Response to Change in Transmission Intensity: A Review of the Current State of Research

Malaria parasites can adjust the proportion of parasites that develop into gametocytes, and thus the probability for human-to-vector transmission, through changes in the gametocyte conversion rate. Understanding the factors that impact the commitment of malaria parasites to transmission is required to design better control interventions. Plasmodium spp. persist across countries with vast differences in transmission intensities, and in sites where transmission is highly seasonal. Mounting evidence shows that Plasmodium spp. adjusts the investment in transmission according to seasonality of vector abundance, and transmission intensity. Various techniques to determine the investment in transmission are available, i.e., short-term culture, where the conversion rate can be measured most directly, genome and transcriptome studies, quantification of mature gametocytes, and mosquito feeding assays. In sites with seasonal transmission, the proportion of gametocytes, their densities and infectivity are higher during the wet season, when vectors are plentiful. When countries with pronounced differences in transmission intensity were compared, the investment in transmission was higher when transmission was low, thus maximizing the parasite’s chances to be transmitted to mosquitoes. Increased transmissibility of residual infections after a successful reduction of malaria transmission levels need to be considered when designing intervention measures.

Development of an in vitro tissue culture system for hammer coral (Fimbriaphyllia ancora) ovaries

In vitro gonad culture systems have proven useful to investigate intrinsic mechanisms of sexual reproduction in animals. Here we describe development of an in vitro culture method for coral ovaries. Mesenterial tissues containing both ovaries and mesenterial filaments were microscopically isolated from the scleractinian coral, Fimbriaphyllia ancora, and culture conditions were optimized. M199 diluted 10× (10% M199, pH 8.1) and supplemented with 25 mM HEPES and the antibiotics, ampicillin, penicillin and streptomycin, supported oocyte survival and maintained the structural integrity of ovaries during short-term culture (~ 6 days). Addition of a commercial antibiotic–antimycotic solution (Anti–Anti) and fetal bovine serum adversely affected ovary maintenance and caused tissue disintegration. Characterization of cultured ovaries showed that there is no difference in cell proliferation of ovarian somatic cells between culture Days 1 and 6. Moreover, the presence of oogonia and expression of a major yolk protein, vitellogenin, were confirmed in ovaries cultured for 6 days. This system will be useful for studying effects of a wide range of substances on coral oogenesis.

Viability and Infectivity of Plasmodium vivax Gametocytes in Short-Term Culture

The control and elimination of malaria caused by Plasmodium vivax both represent a great challenge due to the biological aspects of the species. Gametocytes are the forms responsible for the transmission of the parasite to the vector and the search for new strategies for blocking transmission are essential in a scenario of control and elimination The challenges in this search in regard to P. vivax mainly stem from the lack of a long-term culture and the limitation of studies of gametocytes. This study evaluated the viability and infectivity of P. vivax gametocytes in short-term culture. The samples enriched in gametocytes using Percoll (i), using magnetic-activated cell sorting (MACS®) (ii), and using non-enriched samples (iii) were evaluated. After the procedures, gametocytes were cultured in IMDM medium for up to 48 h. Cultured P. vivax gametocytes were viable and infectious for up to 48 h, however differences in viability and infectivity were observed in the samples after 12 h of culture in relation to 0 h. Percoll-enriched samples were shown to be viable in culture for longer intervals than those purified using MACS®. Gametocyte viability after enrichment procedures and short-term culture may provide new avenues in the development of methods for evaluating P. vivax TB.

Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry for Identification of Microorganisms in Clinical Urine Specimens after Two Pretreatments

Rapid identification of microorganisms in urine is essential for patients with urinary tract infections (UTIs). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been proposed as a method for the direct identification of urinary pathogens. Our purpose was to compare centrifugation-based MALDI-TOF MS and short-term culture combined with MALDI-TOF MS for the direct identification of pathogens in urine specimens. We collected 965 urine specimens from patients with suspected UTIs, 211/965 isolates were identified as positive by conventional urine culture. Compared with the conventional method, the results of centrifugation-based MALDI-TOF MS were consistent in 159/211 cases (75.4%), of which 135/159 (84.9%) had scores ≥ 2.00; 182/211 cases (86.3%) were detected using short-term culture combined with MALDI-TOF MS, of which 153/182 (84.1%) had scores ≥ 2.00. There were no apparent differences among the three methods (p = 0.135). MALDI-TOF MS appears to accelerate the microbial identification speed in urine and saves at least 24 to 48 hours compared with the routine urine culture. Centrifugation-based MALDI-TOF MS is characterized by faster identification speed; however, it is substantially affected by the number of bacterial colonies. In contrast, short-term culture combined with MALDI-TOF MS has a higher detection rate but a relatively slow identification speed. Combining these characteristics, the two methods may be effective and reliable alternatives to traditional urine culture.

Short term culture of wild caught juvenile rabbit fishes (Siganus javus) in open sea cages at Palk Bay

Objectives: The main objective of the study was to collect and rear the unmarketable, small sized S.javus which is mostly rejected by the traditional fisherman. During fishing activities, under sized S.javus ranging in sizes from 25 to 40 kg (< 30-40 % of total catch/day) is being captured. Due to very low marketable value these small fishes (<250 g) get rejected. Therefore, we utilized this as an economically viable option for utilization as seeds for rearing until they reach the necessary marketable size. Methods: Undersized juveniles (12-16 cm, 40 -55 g) were collected from nearby Olaikuda village,Rameshwaram using traditional traps made using bamboo and odai tree bark (Acacia planifrons). The collected juveniles were stocked in sea cages and fed with locally formulated plant based feed containing 28-32 % crude protein. Findings: Harvesting was successfully done after 140 days with 90 % survival. The average growth rate achieved was 2.5 g /day with FCR of 2.01. This resulted in an average harvest size of 400 g/fish from its initial stoking size of 50 g with an attracted market value of Rs. 150/kg. Novelty: Till date, no attempts were made to commercialize such harvesting techniques for the species Siganus javus in pilot scale in India (Except James, 1984). Keywords: Rabbit fish; Siganus javus; sea cage culture; rabbit fish culture; Siganidae

67 The growth and development of invivo-derived dromedary embryos during short-term incubation: Use of embryo holding medium and the effect of embryonic morphology

Production of invivo embryos for transfer in dromedary camel is a well-established practice, whereas freezing of these embryos is still an ongoing challenge. A common approach in evaluation of freeze–thawing method is achieved by studying invitro development of frozen–thawed embryos. However, not much is known about the development pattern of fresh dromedary embryos during incubation. The objectives of this study were to evaluate the usefulness of commercial holding media for easy short-term culture of these embryos and to provide preliminary insights on the growth and development of hatched blastocysts with different shapes. Recovered hatched blastocysts from superovulated donors were graded as transferable and non-transferable. Embryos with significant folding or crinkliness were further categorized as collapsed, whereas those with a round or oval appearance were categorized as spherical. Culture was performed in 500-μL drops at 38.5°C, 5% O2, 0–6% CO2, and maximum humidity in groups of 2 to 4. The 4 experimental media included culture medium (CM; TCM-199, 10% fetal calf serum (FCS), 0.3mM sodium pyruvate, 2.2mg mL−1 sodium bicarbonate), serum-supplemented holding medium (SSH; Syngro+10% FCS), serum-free holding medium (Syngro) and V-Onestep (Vitromed). In experiment 1, a total of 36 embryos were assigned to 4 groups and further development of the embryos was monitored up to 96h by morphological evaluations, identifying static and degenerating embryos on daily basis. In experiment 2, a total of 16 spherical and 16 collapsed embryos were cultured in SSH and CM and two-thirds of the culture drop was replaced with fresh medium at 72h. The proportion of developing embryos and their size expansion was compared between treatments by Fisher’s test and Mann–Whitney U test, respectively. Statistically similar proportions of embryos continued to develop in all media within the first 48h despite a numeric advantage in CM group; at 72h, the proportion of growing embryos was significantly higher in CM (77.8%) and SSH (66.6%) compared with SFH (11.1%) and OneStep (22.2%) (P&lt;0.05). None of the embryos in SFH and only 1 embryo in the V-Onestep group survived beyond 72h, whereas 3/9 embryos in SSH and 7/9 embryos in CM continued to expand. In experiment 2, the proportion of spherical embryos that developed was higher compared with their collapsed counterparts (8/8 in both groups vs. 5/8 and 4/8 in CM and SSH, respectively) at 24h. However, remaining collapsed embryos grew and expanded at similar rates to spherical ones in each group (P&gt;0.05). Replacing the medium did not favour continuation of embryonic growth in SSH beyond 72 h; only 5/16 embryos survived to 96h compared with 12/16 in CM. In conclusion, serum-supplemented commercial holding preparations provide comparable results to culture medium for short-term incubation of invivo dromedary embryos. Natural collapsing of hatched blastocysts might be associated with lower developmental competence.

The impact of post-warming culture duration on clinical outcomes of vitrified-warmed single blastocyst transfer cycles

Objective: The objective of the study was to compare the effects of long-term and short-term embryo culture to assess whether there is a correlation between culture duration and clinical outcomes. Methods: Embryos were divided into two study groups depending on whether their post-warming culture period was long-term (20–24 hours) or short-term (2–4 hours). Embryo morphology was analyzed with a time-lapse monitoring device to estimate the appropriate timing and parameters for evaluating embryos with high implantation potency in both groups. Propensity score matching was performed to adjust the confounding factors across groups. The grades of embryos and blastoceles, morphokinetic parameters, implantation rate, and ongoing pregnancy rate were compared.Results: No significant differences were observed in the implantation rate or ongoing pregnancy rate between the two groups (56.3% vs. 67.9%, p=0.182; 47.3% vs. 53.6%, p=0.513). After warming, there were more expanded and hatching/hatched blastocysts in the long-term culture group than in the short-term culture group, but there was no significant between-group difference in embryo grade. Regarding pregnancy outcomes, the completion of re-expansion was faster in women who became pregnant than in those who did not for both culture durations (long-term: 2.19±0.63 vs. 4.11±0.81 hours, p=0.003; short-term: 1.17±0.29 vs. 1.94±0.76 hours, p=0.018, respectively). Conclusion: The outcomes of short-term culture and long-term culture were not significantly different in vitrified-warmed blastocyst transfer. Regardless of the post-warming culture time, the degree of blastocyst re-expansion 3–4 hours after warming is an important marker for embryo selection.