Molecular detection of Schistosoma japonicum in infected snails and mouse faeces using a real-time PCR assay with FRET hybridisation probes

A real-time polymerase chain reaction (PCR) assay with fluorescence resonance energy transfer (FRET) hybridisation probes combined with melting curve analysis was developed to detect Schistosoma japonicum in experimentally infected snails and in faecal samples of infected mice. This procedure is based on melting curve analysis of a hybrid between an amplicon from the S. japonicum internal transcribed spacer region 2 sequence, which is a 192-bp S. japonicum-specific sequence, and fluorophore-labelled specific probes. Real-time FRET PCR could detect as little as a single cercaria artificially introduced into a pool of 10 non-infected snails and a single egg inoculated in 100 mg of non-infected mouse faeces. All S. japonicum-infected snails and all faecal samples from infected mice were positive. Non-infected snails, non-infected mouse faeces and genomic DNA from other parasites were negative. This assay is rapid and has potential for epidemiological S. japonicum surveys in snails, intermediate hosts and faecal samples of final hosts.

Trichomonas vaginalis and Tritrichomonas foetus: interaction with fibroblasts and muscle cells - new insights into parasite-mediated host cell cytotoxicity

Trichomonas vaginalis and Tritrichomonas foetus are parasitic, flagellated protists that inhabit the urogenital tract of humans and bovines, respectively. T. vaginalis causes the most prevalent non-viral sexually transmitted disease worldwide and has been associated with an increased risk for human immunodeficiency virus-1 infection in humans. Infections by T. foetus cause significant losses to the beef industry worldwide due to infertility and spontaneous abortion in cows. Several studies have shown a close association between trichomonads and the epithelium of the urogenital tract. However, little is known concerning the interaction of trichomonads with cells from deeper tissues, such as fibroblasts and muscle cells. Published parasite-host cell interaction studies have reported contradictory results regarding the ability of T. foetus and T. vaginalis to interact with and damage cells of different tissues. In this study, parasite-host cell interactions were examined by culturing primary human fibroblasts obtained from abdominal biopsies performed during plastic surgeries with trichomonads. In addition, mouse 3T3 fibroblasts, primary chick embryo myogenic cells and L6 muscle cells were also used as models of target cells. The parasite-host cell cultures were processed for scanning and transmission electron microscopy and were tested for cell viability and cell death. JC-1 staining, which measures mitochondrial membrane potential, was used to determine whether the parasites induced target cell damage. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling staining was used as an indicator of chromatin damage. The colorimetric crystal violet assay was performed to ana-lyse the cytotoxicity induced by the parasite. The results showed that T. foetus and T. vaginalis adhered to and were cytotoxic to both fibroblasts and muscle cells, indicating that trichomonas infection of the connective and muscle tissues is likely to occur; such infections could cause serious risks to the infected host.

Comparison of a modified shell vial culture procedure with conventional mouse inoculation for rabies virus isolation

Rabies is a neurotropic disease that is often lethal. The early diagnosis of rabies infection is important and requires methods that allow for the isolation of the virus from animals and humans. The present study compared a modified shell vial (MSV) procedure using 24-well tissue culture plates with the mouse inoculation test (MIT), which is considered the gold standard for rabies virus isolation. Thirty brain samples (25 positive and 5 negative by the fluorescent antibody test) obtained from different animal species at the National Institute of Hygiene Rafael Rangel in Caracas, Venezuela, were studied by the MIT and MSV assays. Nine samples (36%) were positive at 24 h, 10 (40%) were positive at 48 h and six (24%) were positive at 72 h by the MSV assay. With the MIT assay, 76% were positive at six days post inoculation and 12% were positive at 12 and 18 days post inoculation. One sample that was negative according to the MSV assay was positive with MIT on the 12th day. The MSV procedure exhibited a sensitivity of 96.2%, a specificity of 100%, a positive predictive value of 100% and a negative predictive value 80%. This procedure allowed for rapid rabies virus detection. MIT can be employed as an alternative method in laboratories without tissue culture facilities.

Culture of mouse peritoneal macrophages with mouse serum induces lipid bodies that associate with the parasitophorous vacuole and decrease their microbicidal capacity against Toxoplasma gondii

Lipid bodies [lipid droplets (LBs)] are lipid-rich organelles involved in lipid metabolism, signalling and inflammation. Recent findings suggest a role for LBs in host response to infection; however, the potential functions of this organelle in Toxoplasma gondii infection and how it alters macrophage microbicidal capacity during infection are not well understood. Here, we investigated the role of host LBs in T. gondii infection in mouse peritoneal macrophages in vitro. Macrophages cultured with mouse serum (MS) had higher numbers of LBs than those cultured in foetal bovine serum and can function as a model to study the role of LBs during intracellular pathogen infection. LBs were found in association with the parasitophorous vacuole, suggesting that T. gondii may benefit from this lipid source. Moreover, increased numbers of macrophage LBs correlated with high prostaglandin E2 (PGE2) production and decreased nitric oxide (NO) synthesis. Accordingly, LB-enriched macrophages cultured with MS were less efficient at controlling T. gondii growth. Treatment of macrophages cultured with MS with indomethacin, an inhibitor of PGE2 production, increased the microbicidal capacity against T. gondii. Collectively, these results suggest that culture with MS caused a decrease in microbicidal activity of macrophages against T. gondii by increasing PGE2 while lowering NO production.

Effect of mouse antisera targeting the Phlebotomus papatasi midgut chitinase PpChit1 on sandfly physiology and fitness

In sandflies, the absence of the peritrophic matrix (PM) affects the rate of blood digestion. Also, the kinetics of PM secretion varies according to species. We previously characterised PpChit1, a midgut-specific chitinase secreted in Phlebotomus papatasi (PPIS) that is involved in the maturation of the PM and showed that antibodies against PpChit1 reduce the chitinolytic activity in the midgut of several sandfly species. Here, sandflies were fed on red blood cells reconstituted with naïve or anti-PpChit1 sera and assessed for fitness parameters that included blood digestion, oviposition onset, number of eggs laid, egg bouts, average number of eggs per bout and survival. In PPIS, anti-PpChit1 led to a one-day delay in the onset of egg laying, with flies surviving three days longer compared to the control group. Anti-PpChit1 also had a negative effect on overall ability of flies to lay eggs, as several gravid females from all three species were unable to lay any eggs despite having lived longer than control flies. Whereas the longer survival might be associated with improved haeme scavenging ability by the PM, the inability of females to lay eggs is possibly linked to changes in PM permeability affecting nutrient absorption.

Carbon sources and polyethylene glycol on soybean somatic embryo conversion

Somatic embryogenesis is an efficient method for the production of target cells for soybean genetic transformation. However, this method still offers low percentages of plant regeneration, and perhaps is related to the maturation process and high morphological abnormalities of the matured embryos. This study aimed to identify a maturation medium that could contribute to the outcome of more efficient plant regeneration results. Embryogenic clusters, derived from cotyledons of immature seeds of the soybean cultivars Bragg and IAS5, were used as starting material for embryos development. Different maturation media were tested by using 6% maltose, 3% sucrose or 6% sucrose, combined with or without 25 g L-1 of the osmotic regulator polyethylene glycol (PEG-8000). The histodifferentiated embryos were quantified and classified in morphological types. Percentages of converted embryos were analyzed. Cultivar Bragg resulted in higher matured embryo quantities, but lower percentages were obtained for the conversion in comparison to cultivar IAS5. While the addition of PEG did not affect the number of embryos converted into plants, 6% sucrose enhanced the conversion percent significantly.

Embryo rescue from interspecific crosses in apple rootstocks

The objetive of this work was to rescue immature embryos of apple rootstocks Malus prunifolia (Marubakaido) and Malus pumila (M9) after 40-60 days of pollination and to put them into MS culture media supplemented with agar (6 g L-1) and casein hydrolysate (500 mg L-1). Embryos originated from interspecific crosses and open pollination showed differences in the in vitro responses, depending on the female parent, the developmental stage of the embryo, and the culture medium composition. Embryos of the M. pumila rootstock, rescued within 40 days after pollination and put in culture medium supplemented with indolacetic acid (IAA), gibberellic acid (GA3), kinetin and maltose, resulted in a normal development of plantlets. However, embryos originating from hand-pollination, cultivated in medium supplemented with 14 µM IAA, 5 µM kinetin and 1.5 µM Ga3 (MS1), mainly those of M. prunifolia x M. pumila, showed a high percentage of rusted embryos (96.2%). Embryos from open pollination of M. prunifolia and M. pumila formed calluses. It was possible to identify the influence of the female parent by the enhanced development of M. pumila shoots derived from open or hand-pollination. The crossing of responsive species and the use of the technique of embryo culture provided a rapid and uniform germination and, consequently, the development of fully normal seedlings.

In vitro maintenance, under slow-growth conditions, of oil palm germplasm obtained by embryo rescue

The objective of this work was to evaluate the in vitro maintenance of oil palm (Elaeis guineensis and E. oleifera) accessions under slow-growth conditions. Plants produced by embryo rescue were subject to 1/2MS culture medium supplemented with the carbohydrates sucrose, mannitol, and sorbitol at 1, 2, and 3% under 20 and 25±2ºC. After 12 months, the temperature of 20°C reduced plant growth. Sucrose is the most appropriate carbohydrate for maintaining the quality of the plants, whereas mannitol and sorbitol result in a reduced plant survival.

Assessment of embryo/fetus during pregnancy by threedimensional ultrasonography using the HD live software: iconographic essay

Fetal development is studied since the advent of two-dimensional ultrasonography. However, a detailed assessment of structures and surfaces improved with three-dimensional ultrasonography. Currently, it is possible to identify embryonic components and fetal parts with greater detail, at all pregnancy trimesters, using the HD live software, where the images gain realistic features by means of appropriate control of lighting and shadowing effects. In the present study, the authors utilized this resource to follow-up, by means of images, the development of a normal pregnancy along all trimesters.

Experimentally induced intravaginal Tritrichomonas foetus infection in a mouse model

The interest to develop research on the host-parasite relationship in bovine tritrichomonosis has accomplished the use of experimental models alternative to cattle. The BALB/c mouse became the most appropriate species susceptible to vaginal Tritrichomonas foetus infection requiring previous estrogenization. For the need of an experimental model without persistent estrogenization and with normal estrous cycles, the establishment and persistence of vaginal infection on BALB/c mouse with different concentrations of T. foetus in two experimental groups was evaluated. Group A was treated with 5mg of b-estradiol 3-benzoate to synchronize the estrous, 48 hours before the T. foetus vaginal inoculation, and Group B was inoculated in natural estrus. At 5-7 days after treatment, estrogenic effect decreased allowing all animals to cycle regularly during the experiment. From the first week post-infection, samples of vaginal mucus were taken from all animals during 34 weeks, in order to evaluate the course of infection and the stage of the estrus cycle. Group A showed 93.6% of infected animals, and Group B showed 38%. Different doses of T. foetus were assayed to establish the vaginal infection, with a persistence of 34 weeks. Although different behavior was observed in each subgroup belonging to either Group A or Group B, there were no significant differences among the infecting doses used. The b-estradiol 3-benzoate treatment had a favorable effect on the establishment of the infection (P<0.0001), but it did not influence its persistence (P=0.1097). According to the results, an experimental mouse model is presented, appropriate for further studies on mechanisms of pathogenicity, immune response, protective evaluation of immunogen and therapeutic effect of drugs.

Reproductive efficiency of asymptomatic Theileria equi carriers mares submitted to an embryo transfer program

This study aimed to assess and evaluate the effects of Theileria equi infection on embryonic recovery, gestation and early embryonic loss. Thirteen Mangalarga Marchador Theileria equi positive donors (diagnosed through nested-PCR) and 40 embryos receptors were used. Donors were submitted to two embryo collections in two consecutive estrous cycles (GId); after, the same mares were treated with imidocarb dipropionate (1.2mg/kg IM.) in order to collect more embryos in two more estrous cycles (GIId). Receptors were divided into two groups (control and with treated) with 20 animals each, where one group was the control (GIr) and the other one (GIIr) treated with 1.2mg/kg IM of imidocarb dipropionate assessing the gestation rate at 15, 30, 45 and 60 days. After 52 embryo collections, the embryonic recovery rates were 53.84% (14/26) and 65.38% (17/26) (p> 0.05) for GId and GIId, respectively. The gestation rate was 70% (14/20) (p>0.05) at 15, 30, 45 and 60 days in group GIr and for GIIr was 85% (17/20) (p>0.05) at 15 days, 80% (16/20) (p>0.05) at 30, 45 and 60 days. The treatment with imidocarb dipropionate did not cause significant improvement in the reproductive efficiency at an ET program.

Follicle and corpus luteum size and vascularity as predictors of fertility at the time of artificial insemination and embryo transfer in beef cattle

Abstract:Two ultrasound based fertility prediction methods were tested prior to embryo transfer (ET) and artificial insemination (AI) in cattle. Female bovines were submitted to estrous synchronization prior to ET and AI. Animals were scanned immediately before ET and AI procedure to target follicle and corpus luteum (CL) size and vascularity. In addition, inseminated animals were also scanned eleven days after insemination to target CL size and vascularity. All data was compared with fertility by using gestational diagnosis 35 days after ovulation. Prior to ET, CL vascularity showed a positive correlation with fertility, and no pregnancy occurred in animals with less than 40% of CL vascularity. Prior to AI and also eleven days after AI, no relationship with fertility was seen in all parameters analyzed (follicle and CL size and vascularity), and contrary, cows with CL vascularity greater than 70% exhibit lower fertility. In inseminated animals, follicle size and vascularity was positive related with CL size and vascularity, as shown by the presence of greater CL size and vascularity originated from follicle with also greater size and vascularity. This is the first time that ultrasound based fertility prediction methods were tested prior to ET and AI and showed an application in ET, but not in AI programs. Further studies are needed including hormone profile evaluation to improve conclusion.

Ovule ontogeny of Tabebuia pulcherrima Sandwith (Bignoniaceae): embryo sac development

The chalazal megaspore develops in a Polygonum-type embryo sac. The amyloplast-rich endothelium is partially degraded during the expansion of the micropylar portion of the megagametophyte. Organization of the mature embryo sac is determined by the patterns of vacuolation, nuclear migration, spindle orientation and cellularization. The egg cell is slightly chalazal in relation to the synergids, and its micropylar end does not touch the micropylar channel. At the chalazal pole of the egg apparatus, the common walls between the synergids, the egg and central cells, despite their tenuity, are present in the mature megagametophyte. The polar nuclei do not fuse before fertilization and the antipodals are persistent until the first stages of endosperm formation. The taxonomic significance of some embryological characters for the Bignoniaceae is discussed.

Effect of abscisic acid on the mobilisation of galactomannan and embryo development of Sesbania virgata (Cav.) Pers. (Leguminosae - Faboideae)

Galactomannans (GM) are storage cell wall polysaccharides present in endospermic seeds of legumes. They are thought to be storage polymers, since it has been observed for a few species (among them Sesbania virgata) that they are completely broken down after germination and their products are transferred to the growing embryo. We examined the effect of 10-4 M abscisic acid (ABA) on the degradation of galactomannan in isolated endosperms and intact seeds of S. virgata. We found that after seed germination the initial embryo growth was retarded. Ultrastructural analysis showed that the embryo is completely surrounded by an endosperm which displays very thick galactomannan-containing cell walls. Although an inhibitory effect has been observed on the increase of fresh mass of the embryo, the effect of ABA on the dry mass was weaker and transitory (from 48 to 96 h). Endosperm dry mass and galactomannan degradation were significantly inhibited and the activity of alpha-galactosidase was strongly affected. The addition of ABA before and/or after the start of mobilisation in intact seeds or isolated endosperms, showed that whereas addition before mobilisation did not affect dry mass decrease in intact seeds, it was strongly affected in isolated endosperms. On the other hand, whereas it affected embryo fresh mass increase in intact seeds, but not in isolated embryos, no significant effect was observed on dry mass. These results suggest that ABA affects galactomannan degradation and by doing so, prevents water absorption by the embryo, rather than affect its dry mass. As ABA has been detected in the endosperm of seeds of S. virgata, it is proposed that it probably acts as a modulator of galactomannan mobilisation and consequently synchronises it with early growth of the embryo.

Clastogenic activity of integerrimine determined in mouse micronucleus assays

The pyrrolizidine alkaloid integerrimine, obtained from Senecio brasiliensis, was tested by acute dosing at two concentrations (18.75 and 37.5 mg/kg), and at different times, to establish its ability to induce micronuclei in mouse erythrocytes. This alkaloid was able to increase the frequency of micronucleated polychromatic erythrocytes in both, bone marrow and peripheral blood erythrocytes