Aspectos parasitários observados no local inoculado com esporozoitos de Plasmodium Gallinaceum

The following is a summary of the studies made on the development of Plasmodium gallinaceum sporozoites inoculated into normal chicks. Initially large numbers of laboratory reared Aëdes aegypti were fed on pullets heavily infected with gametocytes. Following the infectious meal the mosquitoes were kept on a diet of sugar and water syrup until the appearance of the sporozoites in the salivary glands. Normal chicks kept in hematophagous arthropod proof cages were then inoculated either by bite of the infected mosquitoes or by subcutaneous inoculations of salivary gland suspensions. By the first method ten mosquitoes fed to engorgement on each normal chick and were then sacrificed immediately afterwards to determine the sporozoite count. By the second method five pairs of salivary glands were dissected out at room temperature, triturated in physiological saline and inoculated subcutaneously. The epidermis and dermis at the site of inoculation were excised from six hours after inoculation to forty eight hours after appearance of the parasites in the blood stream and stretched out on filter paper with the epithelial surface downward. The dermis was then curretted. Slides were made of the scrapings consisting of connective tissue and epithelial cells of the basal layers which were fixed by metyl alcohol and stained with Giemsa for examination under the oil immersion lens. Skin fragments removed from normal chicks and from regions other than the site of inoculation in the infected chicks were used as controls. In these, only the normal histological aspect was ever encountered. In the biopsy made at the earliest period following inoculation clearly defined elongated forms with eight or more chromatin granules arranged in rosary formation were found. The author believes these to be products of the sporozoite evolution. Search for transition stages between these forms and sporozoites is planned in biopsies to be taken immediately following inoculation and at given intervals up to the six hour period. 1.) 6 and 12 hour periods. The bodies referred to above found in the first period in great abundance, apparently in proportion to the large numbers of sporozoites inoculated, were perceptibly reduced in numbers in the second period. 2.) 18 hour period. Only one biopsy was examined. This presented a binuclear body shown in Fig. 1, having a more or less hyaline protoplasm staining an intense blue and a narrow vacuole delimiting the cell boundaries. The two chromatin grains were quite large presenting a clearly defined nuclear texture. 3.) 24 hour period. A similar body to that above (Fig. 2) was seen in the only preparation examined. 4.) 60 hour period. The exoerythrocytic schizonts were found more frequently from this period onward. Several such were found no longer to contain the previously described vacuoles (Fig. 3). 5.) 84 hour period. Cells bearing eight or more schizonts were frequently encountered here. That these are apparently not bodies in process of division may be seen in Fig. 4. From this time onward small violet granules similar to volutine grains appeared constantly in the schizont nucleus and protoplasm. These are definitely not hemozoin. The above observations fell within the incubation period as repeated examinations of the peripheral and visceral blood were negative. Exoery-throcytic parasites also were never encountered in the viscera at this time. Exoerythrocytic schizonts searched for at site of inoculation 1, 24 and 48 hours after the incubation period were present in large number at all three times with apparent tendency to diminish as the number within the blood stream increased. Many of them presented the violet granules mentioned above. The appearance of the chromatin and the intensity of staining of the protoplasm varied from body to body which doubtless corresponds to the evolutionary stage of each. This diversity of aspect may frequently be seen in the parasites of the same host cell (Fig. 5.). These findings lend substance to the theory that the exoerythrocytic forms are the link between the sporozoites and the pigmented parasites of the red blood corpuscles. The explanation of their continued presence in the organism after infection of the blood stream takes place and their presence in cases infected by the inoculation blood does not come within the scope of this work. Large scale observations shortly to be undertaken will be reported in more detail particularly observations on the first evolutionary phases of the sporozoite within the organism of the vertebrate host.

Food availability affects the maternal transfer of androgens and antibodies into eggs of a colonial seabird.

Mothers can improve the quality of their offspring by increasing the level of certain components in their eggs. To examine whether or not mothers increase deposition of such components in eggs as a function of food availability, we food-supplemented black-legged kittiwake females (Rissa tridactyla) before and during egg laying and compared deposition of androgens and antibodies into eggs of first and experimentally induced replacement clutches. Food-supplemented females transferred lower amounts of androgens and antibodies into eggs of induced replacement clutches than did non-food-supplemented mothers, whereas first clutches presented no differences between treatments. Our results suggest that when females are in lower condition, they transfer more androgens and antibodies into eggs to facilitate chick development despite potential long-term costs for juveniles. Females in prime condition may avoid these potential long-term costs because they can provide their chicks with more and higher quality resources.

Culex saltanensis Dyar, 1928: natural vector of Plasmodium juxtanucleare in Rio de Janeiro, Brazil

Searching for the natural vector of Plasmodium juxtanucleare in an enzootic locality: Granjas Calábria (33% of the chickens infected), Jacarepaguá, in Rio de Janeiro, Brazil, 13 comparative captures of mosquitoes were carried out, simultaneously on man (out-doors) and on chiken (in a poultry-yard), between 6 and 9 p.m., from September to March 1989. Culex saltanensis was the most frequent species in captures on chicken, accounting for 41.7% of the mosquitoes collected on this bait, showing to be highly ornithophilic (90% captured on chicken versus 10% on man). Seven specimens of Cx. saltanensis were found naturally infected in granjas Calábria: five with mature pedunculate oocysts and two with sporozoites (on in the haemocoele and one in the salivary glands). These sporozoites porudced an infection by P. juxtanucleare in a chick, which had parasitemia on day 41 after inoculation. One Cx. coronator was found with mature pedunculate oocysts. Culex saltanensis was regarded as primary vector of P. juxtanucleare in Rio de Janeiro for being highly ornithophilic and in enough density to maintain the transmission, having been found with infective sporozoites in its salivary glands, and being susceptible to the parasite and able to transmit experimentally it by the bite.

Molecular analysis of yellow fever virus 17DD vaccine strain

The Oswaldo Cruz Foundation produces most of the yellow fever (YF) vaccine prepared world wide. As part of a broader approach to determine the genetic variability in YF l7D seeds and vaccines and its relevance to viral attenuation the 17DD virus was purifed directly from chick embryo homogenates which is the source of virus used for vaccination of millions of people in Brazil and other countries for half a century. Neutralization and hemagglutination tests showed that the purified virus is similar to the original stock. Furthermore, radioimmune precipitation of 35S-methionine-labeled viral proteins using mouse hyperimmune ascitic fluid revealed identical patterns for the purified 17DD virus and the YF l7D-204 strain except for the 17DD E protein which migrated slower on SDS-PAGE. This difference is likely to be due to N-linked glycosylation. Finally, comparison by northern blot nybridization of virion RNAs of purified 17DD with two other strains of YF virus only fenome-sized molecules for all three viruses. These observations suggest that vaccine phenotype is primarily associated with the accumulation of mutations.

Differential contribution of mitochondria, NADPH oxidases, and glycolysis to region-specific oxidant stress in the anoxic-reoxygenated embryonic heart.

The ability of the developing myocardium to tolerate oxidative stress during early gestation is an important issue with regard to possible detrimental consequences for the fetus. In the embryonic heart, antioxidant defences are low, whereas glycolytic flux is high. The pro- and antioxidant mechanisms and their dependency on glucose metabolism remain to be explored. Isolated hearts of 4-day-old chick embryos were exposed to normoxia (30 min), anoxia (30 min), and hyperoxic reoxygenation (60 min). The time course of ROS production in the whole heart and in the atria, ventricle, and outflow tract was established using lucigenin-enhanced chemiluminescence. Cardiac rhythm, conduction, and arrhythmias were determined. The activity of superoxide dismutase, catalase, gutathione reductase, and glutathione peroxidase as well as the content of reduced and oxidized glutathione were measured. The relative contribution of the ROS-generating systems was assessed by inhibition of mitochondrial complexes I and III (rotenone and myxothiazol), NADPH oxidases (diphenylene iodonium and apocynine), and nitric oxide synthases (N-monomethyl-l-arginine and N-iminoethyl-l-ornithine). The effects of glycolysis inhibition (iodoacetate), glucose deprivation, glycogen depletion, and lactate accumulation were also investigated. In untreated hearts, ROS production peaked at 10.8 ± 3.3, 9 ± 0.8, and 4.8 ± 0.4 min (means ± SD; n = 4) of reoxygenation in the atria, ventricle, and outflow tract, respectively, and was associated with arrhythmias. Functional recovery was complete after 30-40 min. At reoxygenation, 1) the respiratory chain and NADPH oxidases were the main sources of ROS in the atria and outflow tract, respectively; 2) glucose deprivation decreased, whereas glycogen depletion increased, oxidative stress; 3) lactate worsened oxidant stress via NADPH oxidase activation; 4) glycolysis blockade enhanced ROS production; 5) no nitrosative stress was detectable; and 6) the glutathione redox cycle appeared to be a major antioxidant system. Thus, the glycolytic pathway plays a predominant role in reoxygenation-induced oxidative stress during early cardiogenesis. The relative contribution of mitochondria and extramitochondrial systems to ROS generation varies from one region to another and throughout reoxygenation.

The L-Type Ca+ and KATP channels may contribute to pacing-induced protection against anoxia-reoxygenation in the embryonic heart model.

L-Type Ca(2+) and K(ATP) Channels in Pacing-Induced Cardioprotection. AIMS: The L-type Ca(2+) channel, the sarcolemmal (sarcK(ATP)), and mitochondrial K(ATP) (mitoK(ATP)) channels are involved in myocardial preconditioning. We aimed at determining to what extent these channels can also participate in pacing-induced cardioprotection. METHODS: Hearts of 4-day-old chick embryos were paced in ovo during 12 hour using asynchronous intermittent ventricular stimulation at 110% of the intrinsic rate. Sham operated and paced hearts were then submitted in vitro to anoxia (30 minutes) and reoxygenation (60 minutes). These hearts were exposed to L-type Ca(2+) channel agonist Bay-K-8644 (BAY-K) or blocker verapamil, nonselective K(ATP) channel antagonist glibenclamide (GLIB), mitoK(ATP) channel agonist diazoxide (DIAZO), or antagonist 5-hydroxydecanoate. Electrocardiogram, electromechanical delay (EMD) reflecting excitation-contraction (E-C) coupling, and contractility were determined. RESULTS: Under normoxia, heart rate, QT duration, conduction, EMD, and ventricular shortening were similar in sham and paced hearts. During reoxygenation, arrhythmias ceased earlier and ventricular EMD recovered faster in paced hearts than in sham hearts. In sham hearts, BAY-K (but not verapamil), DIAZO (but not 5-hydroxydecanoate) or GLIB accelerated recovery of ventricular EMD, reproducing the pacing-induced protection. By contrast, none of these agents further ameliorated recovery of the paced hearts. CONCLUSION: The protective effect of chronic asynchronous pacing at near physiological rate on ventricular E-C coupling appears to be associated with subtle activation of L-type Ca(2+) channel, inhibition of sarcK(ATP) channel, and/or opening of mitoK(ATP) channel.

Expression of substance P and preprotachykinin mRNA by primary sensory neurons in culture: regulation by factors present in peripheral and central target tissues.

Primary sensory neurons display various neuronal phenotypes which may be influenced by factors present in central or peripheral targets. In the case of DRG cells expressing substance P (SP), the influence of peripheral or central targets was tested on the neuronal expression of this neuropeptide. DRG cells were cultured from chick embryo at E6 or E10 (before or after establishment of functional connections with targets). Preprotachykinin mRNA was visualized in DRG cell cultures by either Northern blot or in situ hybridization using an antisense labeled riboprobe, while the neuropeptide SP was detected by immunostaining with a monoclonal antibody. In DRG cell cultures from E10, only 60% of neurons expressed SP. In contrast, DRG cell cultures performed at E6 showed a significant hybridization signal and SP-like immunoreactivity in virtually all the neurons (98%). The addition of extracts from muscle, skin, brain or spinal cord to DRG cells cultured at E6 reduced by 20% the percentage of neurons which express preprotachykinin mRNA and SP-like immunoreactivity. Our results indicate that factors issued from targets inhibit SP-expression by a subset of primary sensory neurons and act on the transcriptional control of preprotachykinin gene.

Biological characterization of clones derived from the edmonston strain of measles virus in comparison with schwarz and CAM-70 vaccine strains

Four virus clones were derived from the Edmonston strain of measles virus by repeated plaque purification. These clones were compared with the vaccine strains Schwarz and CAM-70 in terms of biological activities including plaque formation, hemagglutination, hemolysis and replication in Vero cells and chick embryo fibroblasts (CEF). Two clones of intermediate plaque yielded mixed plaque populations on subcultivation whereas the other two, showing small and large plaque sizes, showed stable plaque phenotypes. The vaccine strains showed consistent homogeneous plaque populations. All the Edmonston clones showed agglutination of monkey erythrocytes in isotonic solution while both vaccine strains hemagglutinated only in the presence of high salt concentrations. Variation in the hemolytic activity was observed among the four clones but no hemolytic activity was detected for the vaccine virus strains. Vaccine strains replicated efficiently both in Vero cells and CEF. All four clones showed efficient replication in Vero cells but different replication profiles in CEF. Two of them replicated efficiently, one was of intermediate efficiency and the other showed no replication in CEF. Two of the clones showed characteristics similar to vaccine strains. One in terms of size and homogeneity of plaques, the other for a low hemolytic activity and both for the efficiency of propagation in CEF.

No experimental evidence that sibling competition induces young to switch nests in the colonial Alpine swift, Apus melba

Adoption is frequent in colonial animals where opportunities for dependent young to receive care from nonbiological parents are high. The departure of dependent young from their original family to seek adoption in neighbouring families is thought to be induced by sibling competition for access to limited resources provided by poor-quality parents. We tested this hypothesis in the colonial Alpine swift by manipulating the number of young reared per brood, with the prediction that offspring from enlarged broods switch nests more frequently than those from reduced broods. Although nestling swifts hatch with little locomotor activity, from 20 days until their first flight at 50-70 days they frequently move out of their nests to seek adoption in neighbouring families. Although nestlings reared in experimentally enlarged broods were lighter and their body mass at day 20 after hatching was more variable than in nestlings reared in reduced broods, there was no difference between the two treatments in the frequency of nests switching and in the age when nestlings switched nests for the first time. However, consistent with other evidence that nest switching by nestling swifts evolved as a strategy to reduce ectoparasite load, young from broods with naturally high numbers of the ectoparasitic louse fly Crataerina melbae were more prone to switch nests. This shows that ectoparasitism rather than sibling competition is a key proximate factor promoting the evolution of nest switching in the colonial Alpine swift. (c) 2006 The Association for the Study of Animal Behaviour Published by Elsevier Ltd. All rights reserved.

Rearing and Colonization of Lutzomyia evansi (Diptera: Psychodidae), a Vector of Visceral Leishmaniasis in Colombia

The sandfly Lutzomyia evansi from a focus of visceral leishmaniasis in northern Colombia was reared and maintained under laboratory conditions for five generations. The average time for total development was 41.8 days (range = 35.1- 49.6) at 25 oC and 89-95% of relative humidity. The mean number of eggs laid was lower in laboratory bred females either in pots (13.2 eggs/female) or vials (29.9 eggs/female) than in wild caught females (33.4 eggs/female). Immature mortality, mainly due to fungal and mite contamination, was higher during the first two instars than in the remaining immature stages. Adults were robust and healthy although difficult to feed on hamster or chick skin membrane. In summary, Lu. evansi is a colonizable species but requires specific conditions.

Synergistic effect of GDNF and NGF on axonal branching and elongation in vitro.

There is a clinical need to enhance functional recovery of injured peripheral nerves. Local administration of neurotrophic factors (NTFs) after surgical repair has been proposed for this purpose. Little is known, however, on the optimal local dose and dosing frequency of NTFs in a peripheral nerve defect. For increasing our knowledge on biologically relevant local NTFs concentrations and for making available an in vitro assay for assessing the bioactivity of NTFs in connection with implantable localized delivery systems, we developed in this study a bioassay for NTFs, which is based on dorsal root ganglion (DRG) explants from E9 (9 days old) chicken embryos. Axonal elongation and extent of axonal branching was analyzed microscopically after addition of glial cell line-derived neurotrophic factor (GDNF) and nerve growth factor (NGF), each alone and in combination. GDNF significantly promoted axonal elongation, but only little axonal branching, whereas NGF induced extensive axonal branching with modest axonal elongation. The combination of GDNF and NGF exerted a synergistic effect on the axonal elongation, axonal branching and growth kinetics. GDNF and NGF also enhanced the expression of their respective functional receptors Ret and TrkA on the DRG neurons. This information should be relevant for the development of implants containing NTFs and on drug therapy of damaged peripheral nerves.

Involvement of CD73, equilibrative nucleoside transporters and inosine in rhythm and conduction disturbances mediated by adenosine A1 and A2A receptors in the developing heart.

We previously established that exogenous adenosine (ADO) induces transient arrhythmias in the developing heart via the adenosine A1 receptor (A1AR) and downstream activation of NADPH oxidase/ERK and PLC/PKC pathways. Here, we investigated the mechanisms by which accumulation of endogenous ADO and its derived compound inosine (INO) in the interstitial compartment induce rhythm and conduction troubles. The validated model of the spontaneously beating heart obtained from 4-day-old chick embryos was used. Quantitative RT-PCR showed that enzymes involved in ADO and INO metabolism (CD39, CD73 and eADA) as well as equilibrative (ENT1, -3, -4) and concentrative (CNT3) nucleoside transporters were differentially expressed in atria, ventricle and outflow tract. Inactivation of ENTs by dipyridamole, 1) increased myocardial ADO level, 2) provoked atrial arrhythmias and atrio-ventricular blocks (AVB) in 70% of the hearts, 3) prolonged P wave and QT interval without altering contractility, and 4) increased ERK2 phosphorylation. Blockade of CD73-mediated phosphohydrolysis of AMP to ADO, MEK/ERK pathway inhibition or A1AR inhibition prevented these arrhythmias. Exposure to exogenous INO also caused atrial ectopy associated with AVB and ERK2 phosphorylation which were prevented by A1AR or A2AAR antagonists exclusively or by MEK/ERK inhibitor. Inhibition of ADA-mediated conversion of ADO to INO increased myocardial ADO and decreased INO as expected, but slightly augmented heart rate variability without provoking AVB. Thus, during cardiogenesis, disturbances of nucleosides metabolism and transport, can lead to interstitial accumulation of ADO and INO and provoke arrhythmias in an autocrine/paracrine manner through A1AR and A2AAR stimulation and ERK2 activation.

The vigilance components of begging and sibling competition

The conflict between siblings over how parental resources are divided has promoted the evolution of specific behaviour to outcompete each other. Young animals look out for parents' arrival in order to start begging as quickly as possible, since a rapid begging reaction increases the likelihood of being fed before nestmates. If the young can physically intercept the parents, selection might be operating on the offspring ability to monitor parent arrival (vigilance towards parents) and any sudden modifications in siblings' behaviour (vigilance towards siblings). To investigate the adaptive value of nestling vigilance in the context of family interactions, we recorded which direction barn owl Tyto alba siblings were facing in 89 two-chick broods before the first parental feeding visit of the night. Nestlings were more vigilant towards nest entrance than expected by chance suggesting that vigilance towards parents is an important component of sibling competition. When positioned near the nest-box entrance where parents predictably deliver food, the younger individual (i.e. junior) looked more towards the entrance than its older sibling. Thus, when the likelihood of obtaining a food item is relatively high, juniors are more vigilant than seniors to detect the incoming parent. When positioned at the back of the nest, the senior looked relatively more frequently towards its sibling than the junior did in the same situation. This suggests that when the likelihood of obtaining a food item is relatively low, seniors are more vigilant than juniors to observe their sibling. Because vigilance was not related to hunger level and prey obtaining, we propose the hypothesis that vigilance towards parents and siblings only indirectly influences the outcome of sibling competition.

Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data.

Despite the central role of quantitative PCR (qPCR) in the quantification of mRNA transcripts, most analyses of qPCR data are still delegated to the software that comes with the qPCR apparatus. This is especially true for the handling of the fluorescence baseline. This article shows that baseline estimation errors are directly reflected in the observed PCR efficiency values and are thus propagated exponentially in the estimated starting concentrations as well as 'fold-difference' results. Because of the unknown origin and kinetics of the baseline fluorescence, the fluorescence values monitored in the initial cycles of the PCR reaction cannot be used to estimate a useful baseline value. An algorithm that estimates the baseline by reconstructing the log-linear phase downward from the early plateau phase of the PCR reaction was developed and shown to lead to very reproducible PCR efficiency values. PCR efficiency values were determined per sample by fitting a regression line to a subset of data points in the log-linear phase. The variability, as well as the bias, in qPCR results was significantly reduced when the mean of these PCR efficiencies per amplicon was used in the calculation of an estimate of the starting concentration per sample.