Use of an adenosine triphosphate assay, and simultaneous staining with fluorescein diacetate and propidium iodide, to evaluate the effects of cryoprotectants on hard coral (Echinopora spp.) oocytes

The objective was to examine the effects of cryoprotectants on oocytes of hard corals (Echinopora spp.) to obtain basic knowledge for cryopreservation procedures. Oocytes were exposed to various concentrations of cryoprotectants (0.25 to 5.0 M) for 20 min at room temperature (25 °C). Two tests were used to assess ovarian follicle viability: fluorescein diacetate (FDA) + propidium iodide (PI) staining, and adenosine triphosphate (ATP) assay. Both FDA + PI staining and ATP assay indicated that cryoprotectant toxicity to oocytes increased in the order methanol, dimethyl sulfoxide (DMSO), propylene glycol (PG), and ethylene glycol (EG). The no observed effect concentrations for Echinopora spp. oocytes were 1.0, 0.5, 0.25, and 0.25 M for methanol, DMSO, PG, and EG, respectively, when assessed with FDA + PI. The ATP assay was more sensitive than FDA + PI staining (P < 0.05). Oocyte viability after 1.0 M methanol, DMSO, EG, or PG treatment for 20 min at room temperature assessed with FDA + PI tests and ATP assay were 88.9 ± 3.1% and 72.2 ± 4.4%, 66.2 ± 5.0% and 23.2 ± 4.9%, 58.9 ± 5.4% and 1.1 ± 0.7%, and 49.1 ± 5.1% and 0.9 ± 0.5%, respectively. We inferred that the ATP assay was a valuable measure of cellular injury after cryoprotectant incubation. The results of this study provided a basis for development of protocols to cryopreserve coral oocytes.     

Serum Antigen and Antibody Detection in Echinococcosis: Application in Serodiagnosis of Human Hydatidosis

Diagnosis of hydatidosis is based on immunodiagnostic methods along with radiological and ultrasound examinations. The objectives of the present study were to develop a specific and simple antigen-based ELISA method for diagnosis of hydatidosis and compare it with antibody detection method. The subjects in this study included 89 patients in the following groups: surgically confirmed hydatidosis patients (35 cases), control with other parasitic diseases (29 cases), and healthy controls (25 cases). Hyperimmune serum was raised against hydatid cyst fluid in rabbits. Anti-hydatid cyst IgG was purified by affinity chromatography using protein A column and labeled with horseradish peroxidase. Collected sera were assessed for hydatid cyst antigens and antibody by ELISA. Circulating hydatid antigen was found in 9 out of 35 patients with surgically confirmed hydatidosis. A sensitivity of 25.7% and a specificity of 98.0% were calculated for the antigen detection assay. Antibody detection by indirect ELISA, using antigen B, showed that 94.2% of patients (33 cases) have anti-hydatid cyst antibodies in their serum while cross reaction was noted in a few of non-hydatidosis patients. A sensitivity of 94.2% and specificity of 81.6% were found for the antibody detection assay. Findings of this study indicated that antibody detection assay is a sensitive approach for diagnosis of hydatid cyst while antigen detection assay might be a useful approach for assessment of the efficacy of treatment especially after removal of the cyst.     

A solid-phase method for the extraction and measurement of anandamide from multiple human biomatrices

N-Arachidonoylethanolamine (AEA, anandamide) was the first endocannabinoid to be identified and has since become associated with the mediation of several physiological functions and disease states. AEA has been isolated from numerous tissues and biofluids, in the low nanomolar range, using lipid extraction techniques with organic solvents. These techniques require the drying down of relatively large volumes of solvents, making them unsuitable for high-throughput analysis. Here we describe a solid-phase extraction (SPE) method for the investigation of AEA concentrations in human plasma, serum, milk, urine, amniotic fluid, peritoneal fluid, saliva, follicular fluid, and fluid from an ovarian cyst. AEA was detected in serum and plasma from blood isolated from 20 adult women (means ± standard deviations: 0.68 ± 0.29 and 0.64 ± 0.28 nM, respectively), from pregnant women at term (1.37 ± 0.42 nM), and from umbilical vein (1.26 ± 0.33 nM) and umbilical artery (1.14 ± 0.35 nM), in milk (0.12 ± 0.05 nM) and from amniotic (0.03 ± 0.02 nM), peritoneal (0.93 ± 0.27 nM), follicular (1.17 ± 0.51 nM), and ovarian cyst (0.32 ± 0.01 nM) fluids. AEA was undetectable in saliva and urine. The 60% AEA extraction efficiency achieved with SPE from plasma was superior to the 19% efficiency achieved using the existing organic solvent extraction method. Limits of quantification and detection for AEA were also improved dramatically using SPE (8 and 4 fmol/ml) compared with organic extraction (25 and 18.75 fmol/ml plasma). These improvements allow the use of smaller plasma samples with SPE. Intra- and interday variability were comparable, and the mean AEA concentration of pooled plasma samples (1.18 nM, n = 15) was identical with the two techniques. Similarly, when 56 plasma samples from laboring and nonlaboring women were analyzed using both techniques, no extraction method-dependent differences were observed. Consequently, we provide evidence for a robust SPE technique for the extraction of AEA from biomatrices to replace the existing liquid extraction methods, with the SPE technique being superior in terms of speed, extraction efficiency, and sample size required.     

Binax NOW® Streptococcus pneumoniae test of middle ear fluid for detecting causative pathogens in children with acute otitis media

We investigated rapid diagnosis of acute otitis media, (AOM) with the Binax NOW® Streptococcus pneumoniae test kit. Middle ear fluid specimens were obtained from 38 children with AOM (mean age: 1.1 years). Binax NOW® demonstrated 100% sensitivity and 72% specificity, suggesting it is a useful auxiliary test for AOM.     

Efficacy of loop mediated isothermal amplification (LAMP) assay for the laboratory identification of Mycobacterium tuberculosis isolates in a resource limited setting

Current methods of TB diagnosis are time consuming and less suited for developing countries. The LAMP (loop mediated isothermal amplification) is a rapid method more suitable for diagnosis in resource limited settings and has been proposed as a viable test requiring further evaluation for use as a laboratory method as well. We evaluated two LAMP assays, using culture lysates of clinical sputum samples (from Southern India) and compared it to a proprietary multiplex PCR reverse-hybridization line probe assay (‘GenoType MTBC’ from HAIN Lifescience GmbH, Germany). The LAMP procedure was modified to suit the local conditions. The Mycobacterium tuberculosis specific LAMP assay (‘MTB LAMP’) showed sensitivity and specificity, of 44.7% and 94.4% respectively in a 60 min format, 85.7% and 93.9% respectively in a 90 min format and 91.7%, and 90.9% respectively in a 120 min format. The Mycobacteria universal LAMP assay (‘Muniv LAMP’) showed a sensitivity of 99.1%. The LAMP was shown to be a rapid and accessible assay for the laboratory identification of M. tuberculosis isolates. Initial denaturation of template was shown to be essential for amplification in unpurified/dilute samples and longer incubation was shown to increase the sensitivity. The need for modification of protocols to yield better efficacy in this scenario needs to be addressed in subsequent studies.     

Flow cytometric screening for anti-leishmanials in a human macrophage cell line

High-throughput drug screening methods against the intracellular stage of Leishmania have been facilitated by the development of in vitro models of infection. The use of cell lines rather than primary cells facilitates these methods. Peripheral blood mononuclear cell (PBMC) derived macrophages and THP-1 cells were infected with stationary phase egfp transfected Leishmania amazonensis parasites and then treated with anti-leishmanial compounds. Drug activity was measured using a flow cytometric approach, and toxicity was assessed using either the MTT assay or trypan blue dye exclusion. Calculated EC₅₀’s for amphotericin B, sodium stibogluconate, and miltefosine were 0.1445 ± 0.0005 μg/ml, 0.1203 ± 0.018 mg/ml, and 26.71 μM using THP-1 cells, and 0.179 ± 0.035 μg/ml, 0.1948 ± 0.0364 mg/ml, and 13.77 ± 10.74 μM using PBMC derived macrophages, respectively. We conclude that a flow cytometric approach using egfp transfected Leishmania species can be used to evaluate anti-leishmanial compounds against the amastigote stage of the parasite in THP-1 cells with excellent concordance to human PBMC derived macrophages.     

Determination of the optimal culture conditions for detecting thermophilic campylobacters in environmental water

This study evaluated alternative protocols for culturing thermophilic campylobacters in environmental water. All samples were filtered through a sterile 0.45 μm pore-size membrane, which was then incubated in Preston enrichment broth. Four variables were compared: water sample volume (2000 mL vs. 500 mL), enrichment broth volume (25 mL vs. 100 mL), enrichment incubation duration (24 h vs. 48 h), and number of enrichment passages (one vs. two). In addition, DNA extracts were prepared from all final broths and analyzed using three rRNA PCR assays. River water was collected at 3 sampling sites weekly for 9 weeks. Among these 27 collections, 25 (93%) yielded Campylobacter spp. under at least one of the 16 culture conditions. By univariate analysis, yields were significantly better for the 2000 mL sample volume (68.5% vs. 43.0%, p < 0.0001) and the 25 mL enrichment broth volume (64.5% vs. 47.0%, p < 0.0004). Neither of the enrichment period had a significant effect, although there was a trend in favor of 48 h incubation (59.5% vs. 52.0%, p = 0.13). The three PCR methods gave concordant results for 66 (33%) of the culture-negative samples and 103 (50%) of the culture-positive samples. Compared with culture results, Lubeck's 16S PCR assay had the best performance characteristics, with a sensitivity of 82% and a specificity of 94%. Of the 12 culture-negative samples positive by Lubeck's PCR assay, 11 (92%) samples were also positive by Denis' 16S PCR assay, suggesting that in these cases the culture might have been falsely negative. Based on our results, we conclude that the optimal conditions for detecting Campylobacter spp. in natural waters include 2000 mL sample volume and a single enrichment broth of 25 mL PB incubated for 48 h.     

Hepatic growth factor (HGF) inhibits cigarette smoke extract induced apoptosis in human bronchial epithelial cells

Low concentrations of cigarette smoke induced DNA damage and repair without leading to apoptosis in human bronchial epithelial cells. Higher concentrations of cigarette smoke, however, could induce either apoptosis or necrosis. The current study demonstrated that 15% cigarette smoke extract (CSE) induced apoptosis as evidenced by DNA content profiling (17.8 ± 2.1% vs 10.2 ± 1.6% of control, p < 0.05), LIVE/DEAD staining (60.2 ± 2.1% viable cells in CSE-treated vs 86.5 ± 2.3% in control cells, p < 0.05), and COMET assay (24.3 ± 0.6% of Apoptotic Index in the cells treated with CSE vs 4.7 ± 0.6% of control, P < 0.05). Hepatocyte growth factor (HGF) significantly blocked the cigarette smoke-induced apoptosis as shown by DNA profiling (10.8 ± 1.5% of CSE + HGF, p < 0.05), LIVE/DEAD staining (78.5 ± 1.2% in CSE + HGF treated cells, p < 0.05), and COMET assay (Apoptotic Index: 10.0 ± 0.8% in CSE + HGF treated cells, P < 0.05). This protective effect of HGF on CSE-induced apoptosis was abolished by PI3K inhibitors, wortmannin and LY294002, and by introduction of the dominant negative AKT into the cells. Furthermore, CSE plus HGF could induce phosphorylation of AKT Thr 308 and the pro-apoptotic protein, BAD. These results suggest that HGF modulates cell survival in response to cigarette smoke exposure through the PI3K/AKT signaling pathway.     

Added Value of Antigen ELISA in the Diagnosis of Neurocysticercosis in Resource Poor Settings

Background

Neurocysticercosis (NCC) is the most common cause of acquired epilepsy in Taenia solium endemic areas, primarily situated in low-income countries. Diagnosis is largely based upon the “Del Brutto diagnostic criteria” using the definitive/probable/no NCC diagnosis approach. Neuroimaging and specific T. solium cysticercosis antibody detection results are at the mainstay of this diagnosis, while antigen detection in serum has never been included. This study aimed at evaluating the addition of antigen detection as a major diagnostic criterion, especially in areas where neuroimaging is absent.

Methods

The B158/B60 monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for the detection of circulating cysticercus antigen was carried out retrospectively on serum samples collected during a hospital-based study from 83 people with epilepsy (PWE) in an endemic area.

Results

The addition of antigen results as a major criterion allowed the correct diagnosis of definitive NCC in 10 out of 17 patients as opposed to 0/17 without antigen results in the absence of neuroimaging. A sensitivity of 100% and a specificity of 84% were determined for the diagnosis of active NCC using antigen ELISA. While the use of a higher cutoff improves the specificity of the test to 96%, it decreases its sensitivity to 83%.

Conclusions

In areas where neuroimaging is absent, NCC diagnosis according to the existing criteria is problematic. Taking into account its limitations for diagnosis of inactive NCC, antigen detection can be of added value for diagnosing NCC in PWE by supporting diagnostic and treatment decisions. Therefore, we recommend a revision of the “Del Brutto diagnostic criteria” for use in resource poor areas and suggest the inclusion of serum antigen detection as a major criterion.     

Fasciola gigantica: Immunodiagnosis of fasciolosis by detection of circulating 28.5 kDa tegumental antigen

A monoclonal antibody (MoAb)-based sandwich ELISA was developed for the detection of circulating 28.5 kDa tegumental antigen (28.5 kDa TA) in the sera from mice experimentally infected with Fasciola gigantica. The MoAb was immobilized on a microtiter plate, and the antigen in the serum was captured and detected with biotinylated polyclonal rabbit anti TA antibody. The test could detect 28.5 kDa in the extracts of tegument (TA), whole body (WB) and excretory-secretory (ES) fractions at the concentrations of these crude antigens as low as 600 pg/ml, 16 and 60 ng/ml, respectively. This sandwich ELISA assay could detect the infection from day 1 to 35 post infection and showed that circulating level of 28.5 kDa TA peaked at day 1 post infection. In contrast, the antibody detection by indirect ELISA could only demonstrate the antibody level from 35 days post infection. The reliability of the assay method was evaluated using sera from mice infected with F. gigantica or Schistosoma mansoni, and hamsters infected with Opisthorchis viverrini, as well as healthy mice and hamsters. The sandwich ELISA exhibited a sensitivity and specificity at 94.55% and 100%, respectively, and with a positive predictive value of 100%, a negative predictive value of 97.39%, false positive rate of 0%, false negative rate of 5.50% and an accuracy of 98.2%. Thus, this detection method exhibited high specificity and sensitivity as well as could be used for early diagnosis of fasciolosis by F. gigantica.     

An unusual thermostable aspartic protease from the latex of Ficus racemosa (L.)

The most extensively studied ficins have been isolated from the latex of Ficus glabrata and Ficus carica. However the proteases (ficins) from other species are less known. The purification and characterization of a protease from the latex of Ficus racemosa is reported. The enzyme purified to homogeneity is a single polypeptide chain of molecular weight of 44,500 ± 500 Da as determined by MALDI-TOF. The enzyme exhibited a broad spectrum of pH optima between pH 4.5-6.5 and showed maximum activity at 60 ± 0.5 °C. The enzyme activity was completely inhibited by pepstatin-A indicating that the purified enzyme is an aspartic protease. Far-UV circular dichroic spectra revealed that the purified enzyme contains predominantly β-structures. The purified protease is thermostable. The apparent T_m, (mid point of thermal inactivation) was found to be 70 ± 0.5 °C. Thermal inactivation was found to follow first order kinetics at pH 5.5. Activation energy (E_a) was found to be 44.0 ± 0.3 kcal mol⁻¹. The activation enthalpy (ΔH^∗), free energy change (ΔG^∗) and entropy (ΔS^∗) were estimated to be 43 ± 4 kcal mol⁻¹, −26 ± 3 kcal mol⁻¹ and 204 ± 10 cal mol⁻¹ K⁻¹, respectively. Its enzymatic specificity studied using oxidized B chain of insulin indicates that the protease preferably hydrolyzed peptide bonds C-terminal to glutamate, leucine and phenylalanine (at P₁ position). The broad specificity, pH optima and elevated thermal stability indicate the protease is distinct from other known ficins and would find applications in many sectors for its unique properties.     

Echinococcus multilocularis: Single hepatic lesion experimentally established without metastasis in rats

We herein describe the establishment of single hepatic lesions of Echinococcus multilocularis in rats. A 3 mm incision was made on the liver with a surgical knife, and one small round vesicle of E. multilocularis (between 1 × 1 mm and <2 × 2 mm in diameter) was transplanted into the incision and covered with absorbable hemostat gauze. The presence and growth of the transplanted vesicle was monitored for 12 weeks using magnetic resonance imaging (MRI). Hepatic lesions, the metacestode of this parasite were confirmed in 12 of 17 infected rats (70.6%) by MRI and macroscopic examinations. The average size of the metacestodes with brood capsules at 12 weeks after the experimental transplantation of a single vesicle was 6.1 ± 2.5 mm × 4.4 ± 1.5 mm. The smallest size of the metacestodes detected by MRI was approximately 3 × 3 mm. This new approach of establishing single hepatic metacestodes of E. multilocularis in experimental animals is expected to be useful for analyzing the immune-pathological mechanisms of hepatic AE.     

Semen characteristics and sperm morphology of serow (Capricornis sumatraensis)

The serow (Capricornis sumatraensis) is a critically endangered species. The objectives of this study were to evaluate ejaculate quality in captive males, and to investigate and characterize sperm morphology. Semen was collected using electroejaculation. Mean (±S.D.) seminal characteristics were: semen volume 2.3 ± 0.8 mL, pH 7.8 ± 0.4, and osmolality 329.9 ± 32.9 mOsmol/kg; sperm concentration 515.8 ± 263.1 × 10⁶ cells/mL; wave motion score (1-5) 3.9 ± 0.4; motile sperm 60.5 ± 22%; viable sperm 68.3 ± 9.4%; morphologically normal sperm 70.8 ± 19.3%; and an opacity that was yellowish to milky-white. Sperm head length, width, degree of elongation, area, and perimeter were 6.0 ± 0.6 μm, 4.3 ± 0.3 μm, 71.7 ± 8.6%, 19.8 ± 2.5 μm², and 17.9 ± 2.1 μm. Based on these measurements, we categorized sperm head morphometry as small, medium, or large. In addition, sperm morphology was examined by light and scanning electron microscopy; overall, morphologically normal and abnormal sperm were similar to those reported for other bovidae. In summary, this study provided baseline data regarding semen characteristics of C. sumatraensis, which should be of value in the preservation of this endangered species.     

Quantitative analysis of five sterols in amniotic fluid by GC–MS: Application to the diagnosis of cholesterol biosynthesis defects

Cholesterol and its precursors, namely 7-dehydrocholesterol, desmosterol and lathosterol are important biochemical markers of cholesterol biosynthesis, and their quantification in body fluids is useful for the diagnosis of cholesterol biosynthesis pathway disorders. A rapid and sensitive gas chromatographic–mass spectrometric method was developed and validated for quantitative analysis of five sterols (cholesterol, 7-dehydrocholesterol, desmosterol, lathosterol and sitosterol) in amniotic fluid. The method was linear for all compounds (r² > 0.99), and intra and inter-assay coefficients of variation were typically below 5%, and inaccuracy was within a ±12% interval. The method was applied to 330 amniotic fluid samples, grouped by gestational age between 13 and 22 weeks of pregnancy, in order to establish reference intervals for sterols in this specimen. The obtained concentrations (μmol/L) for each sterol was as follows: 22.1758 ± 4.2716 at 13 weeks and 78.5082 ± 12.9041 at 22 weeks for cholesterol; 0.0039 ± 0.0007 at 13 weeks and 0.1150 ± 0.0212 at 22 weeks for 7-dehydrocholesterol; 0.1562 ± 0.0406 at 13 weeks and 0.7691 ± 0.0821 at 22 weeks for desmosterol; 0.0272 ± 0.0035 at 13 weeks and 0.8551 ± 0.1791 at 22 weeks for lathosterol; and 0.0404 ± 0.0039 at 13 weeks and 0.2326 ± 0.0386 at 22 weeks for sitosterol. The method was also applied to one pathological sample that showed decreased levels of cholesterol, and higher concentration of 7-dehydrocholesterol, which is consistent with a 7-dehydrocholesterol-reductase deficiency. Our results showed that as long as pregnancy goes on, the concentrations of cholesterol and precursors increase in amniotic fluid, which is related to the increased need for cholesterol by the fetus. The reference range of each sterol in amniotic fluid was calculated at different gestational ages and will be useful for the interpretation and validation of biochemical prenatal diagnosis of inborn errors of sterol biosynthesis.     

The effects of intertidal air exposure on the respiratory physiology and the killing activity of hemocytes in the pacific oyster, Crassostrea gigas (Thunberg)

The occurrence of summer mortalities of the commercially important Pacific oyster, Crassostrea gigas, has increased in recent years. These mortality events occur during the late summer when water temperatures are at their highest. Many theories have been proposed concerning the causes including reproductive stress, environmental stress, disease, or synergistic interactions of these factors. C. gigas are grown intertidally and are exposed to the air (emersed) for hours at a time. These organisms can experience extreme changes in temperature during the course of a day. An oyster closed during emersion depletes the oxygen stores to near zero within the shell and builds up CO₂ causing a decrease in tissue pH. The focus of this study is to determine the respiratory (pH, Po₂, Pco₂ and total CO₂) and immune responses of oysters exposed to air at normal seasonal temperatures, and to determine whether these stresses associated with emersion inhibit the immune system of the oyster and contribute to the summer mortalities. The respiratory variables of the hemolymph of oysters submerged at 18 °C (pH = 7.52 ± 0.04 S.E.M., Po₂ = 7.09 ± 0.53 S.E.M. kPa and Pco₂ = 0.20 ± 0.03 S.E.M. kPa) varied significantly from oysters emersed for four hours at 22°C (pH = 7.11 ± 0.03 S.E.M., Po₂ = 3.83 ± 0.15 S.E.M. kPa, Pco₂ = 0.36 ± 0.03 S.E.M. kPa) and those emersed for four hours at 30 °C (pH = 6.84 ± 0.02 S.E.M., Po₂ = 3.10 ± 0.12 S.E.M. kPa, Pco₂ = 1.31 ± 0.06 S.E.M. kPa). The ability of hemocytes to kill the bacterium Vibrio campbellii was assessed using an in vitro assay to generate a killing index. There was no significant difference in the killing index between pH treatment groups (p = 0.856): at pH 7.6 killing index = 50.2% ± 2.33 S.E.M., at pH 6.6 killing index = 52.3% ± 3.67 S.E.M.. Temperature was the only factor to significantly affect the killing indices among temperature and oxygen treatment groups. The killing index was lowest (29.3% ± 3.25 S.E.M.) at 30 °C and 7% oxygen, simulating in vivo oxygen pressure in well-aerated conditions and 30 °C and 3% oxygen, simulating in vivo oxygen pressure in hypoxia (30.5% ± 3.25 S.E.M.), compared with the index in 7% oxygen at low temperature (18 °C) (44.4% ± 4.50 S.E.M.) or compared with low oxygen (3%) at low temperature (18 °C) (39.7% ± 2.51 S.E.M.). The seasonal and diurnal rise in temperature may, therefore, be an important factor contributing to summer mortalities of C. gigas.     

Cryobanking as tool for conservation of biodiversity: effect of brown trout sperm cryopreservation on the male genetic potential

Sperm cryobanking could be a good alternative to breeding in captivity in order to preserve genetic diversity. Sperm from two well-characterized brown trout populations originating from two river basins in the Northwest of Spain (Esla and Duerna), both threatened by extinction, was cryopreserved. In order to determine whether a sperm cryobank is the best option for preserving genetic profiles, cell viability, chromatin fragmentation, fertility and genetic variability of the offspring obtained with fresh and frozen sperm, were analyzed. Sperm viability was not reduced by freezing (87.0 ± 3.32% to 77.9 ± 3.59% and 77.6 ± 6.53% to 76.6 ± 2.61% in fresh and frozen sperm from Esla and Duerna, respectively). The percentage of fragmented DNA increased after freezing in spermatozoa from Esla males (from 4.7 ± 0.23% to 6.0 ± 0.28%), but not those from Duerna males.After freezing/thawing, the percentage of eyed embryos drops from 66.8 ± 6.77% to 16.1 ± 3.46% and from 50 ± 8.97% to 11.5 ± 2.50% in the Esla and Duerna basins, respectively. This reduction indicates that many spermatozoa have lost their ability to contribute to embryo development and this loss is not related to either spermatozoa viability or the DNA integrity. Genotypic determination by microsatellite analysis showed that frozen/thawed sperm provided offspring with a similar genetic profile to unfrozen milt, demonstrating the accuracy of the cryopreservation procedure.Taking into account the prolificacy of this species, even a low rate of success of fry after cryopreservation, could provide enough individuals to recover stable populations without altering the genetic profiles of the preserved strains. Therefore, cryopreservation is considered a safe, simple and cheap technology for gene banking in the analyzed brown trout populations.     

Host species diversity and post-blood feeding carbohydrate availability enhance survival of females and fecundity in Aedes albopictus (Diptera: Culicidae)

Aedes albopictus mosquito is an opportunistic blood feeder and has a broad host range. The feeding behavior and habits of this mosquito are liable to increase the transmission potential of arboviruses. The survival and fecundity in A. albopictus fed on different hosts and post-blood meal provision of sugar were investigated in a laboratory-reared colony. Adult survival of caged female A. albopictus that were fed on blood of two different hosts (double meal) was higher than the females fed only on one host (single meal) (mean survival: 70.2 ± 9.6 vs. 55.5 ± 5.5%, respectively) when held in the laboratory for 72 h after blood feeding. Mean survival of females provided 10% sucrose solution (in water) after a single or double blood meal was higher (90.5 ± 6.4% and 89.3 ± 6.5%, respectively) than in the respective groups receiving water only following blood feeding (double meal: 49.0 ± 9.6%; single meal: 45.3 ± 10.9%). Females receiving a double meal were more fecund on average (89.0 ± 6.6 eggs) than females provided a single meal (82.3 ± 8.2 eggs).     

Modelling the age variation of larval protoscoleces of Echinococcus granulosus in sheep

In autumn 2006, a study of the age-dynamics of Echinococcus granulosus cyst abundance was undertaken from an abattoir study of 1081 sheep slaughtered in Naryn Province in central Kyrgyzstan, an area endemic for echinococcosis. The results demonstrated approximately 64% of sheep were infected with the prevalence increasing markedly with age. The mean abundance was 3.8 cysts per sheep. From established models, an infection pressure of 1.33 cysts per year was estimated. In addition all cysts were recovered from infected sheep and the numbers of protoscoleces was evaluated in each cyst. A new model was developed that examined the variation in numbers of protoscoleces per infected sheep with age. This demonstrated that young sheep aged 1-2 years had very few protoscoleces, but there was a massive increase as the sheep aged. The best-fitting model assumed that the number of protoscoleces in a sheep was proportional to the volume of the cysts. In this model, the radius of the individual cyst increased linearly with the age of the cyst and hence the volume increased with the cube of the cyst age. This combined with the linear increase in numbers of cysts with age resulted in a massive accumulation of protoscoleces with the age of sheep. When the model was parameterised it demonstrated that 80% of protoscoleces were present in sheep aged 4 years and older and this represented just 28% sheep slaughtered. An average sheep at 6 or more years of age has an abundance of over 9700 protoscoleces, whilst in a young sheep of 1 year of age an average of just 16 protoscoleces could be found. The average for the sampled population across all ages was 1562 protoscoleces per sheep. The maximum number of protoscoleces in a single cyst was just 482 for sheep aged 1 year rising to 92,000 for sheep aged 6 years or older. The mean volume of cysts containing protoscoleces increased from approximately 0.7 ml at 1 year of age to 8.8 ml at 6 years of age. Cysts containing protoscoleces ranged from a diameter of 0.5-8 cm with a volume of fluid ranging from 0.2 to 50 ml. It is hypothesised that removal of old sheep through a culling programme could substantially improve the control of cystic echinococcosis.     

Genetic detection and quantification of Nosema apis and N. ceranae in the honey bee

The incidence of nosemosis has increased in recent years due to an emerging infestation of Nosema ceranae in managed honey bee populations in much of the world. A real-time PCR assay was developed to facilitate detection and quantification of both Nosema apis and N. ceranae in both single bee and pooled samples. The assay is a multiplexed reaction in which both species are detected and quantified in a single reaction. The assay is highly sensitive and can detect single copies of the target sequence. Real-time PCR results were calibrated to spore counts generated by standard microscopy procedures. The assay was used to assess bees from commercial apiaries sampled in November 2008 and March 2009. Bees from each colony were pooled. A large amount of variation among colonies was evident, signifying the need to examine large numbers of colonies. Due to sampling constraints, a subset of colonies (from five apiaries) was sampled in both seasons. In November, N. apis levels were 1212 ± 148 spores/bee and N. ceranae levels were 51,073 ± 31,155 spores/bee. In March, no N. apis was detected, N. ceranae levels were 11,824 ± 6304 spores/bee. Changes in N. ceranae levels were evident among apiaries, some increasing and other decreasing. This demonstrates the need for thorough sampling of apiaries and the need for a rapid test for both detection and quantification of both Nosema spp. This assay provides the opportunity for detailed study of disease resistance, infection kinetics, and improvement of disease management practices for honey bees.     

Cryopreservation of sperm of an indigenous endangered fish species Nandus nandus (Hamilton, 1822) for ex-situ conservation

This study dealt with the development of cryopreservation protocol for Nandus nandus, which entailed a number of experiments. Sperm was collected by sacrificing males. The collected sperm was suspended in extenders. Activation of sperm motility was evaluated in different osmolalities of NaCl. Motility of sperm decreased as the osmolality of the extender increased and was completely inhibited at almost 319 mOsmol/kg. To evaluate the toxicity of cryoprotectant, sperm was incubated with DMSO, methanol and ethanol at 5%, 10% and 15% concentrations, respectively, for 5–35 min. Five and ten percent of cryoprotectants produced better motility during 5 and 10 min incubation. Sperm incubated with 15% cryoprotectant seemed to be toxic and this concentration was excluded in the subsequent trials. Three extenders, namely, Alsever’s solution, egg-yolk citrate and urea egg-yolk and three cryoprotectants, DMSO, methanol and ethanol were employed to preserve the sperm. Alsever’s solution with 10% DMSO showed best performance producing 90.0 ± 1.8% and 75.0 ± 2.5% equilibration and post-thaw motility followed by that of 82.5 ± 4.2% and 62.5 ± 5.5% with Alsever’s solution plus methanol, respectively. Between two diluents, sperm preserved with Alsever’s solution plus DMSO produced highest fertilization (76.7 ± 3.3%) and hatching (43.8 ± 7.9%) while fresh sperm yielded 83.3 ± 6.7% and 64.0 ± 10.4% fertilization and hatching, respectively. The protocol developed through the study can be applied for long-term conservation of genetic materials of the endangered fish N. nandus and the cryopreserved sperm can be used in artificial breeding for generating new individuals.