The Synaptic Leap

Antibodies play a central role in prophylaxis against many infectious agents. While neutralization is a primary function of antibodies, the Fc- and complement-dependent activities of these multifunctional proteins may also be critical in their ability to provide protection against most viruses. Protection against viral pathogens in vivo is complex, and while virus neutralization—the ability of antibody to inactivate virus infectivity, often measured in vitro—is important, it is often only a partial contributor in protection. The rapid fluorescent focus inhibition test (RFFIT) remains the “gold standard” assay to measure rabies virus–neutralizing antibodies. In addition to neutralization, the rabies-specific antigen-binding activity of antibodies may be measured through enzyme-linked immunosorbent assays (ELISAs), as well as other available methods. For any disease, in selecting the appropriate assay(s) to use to assess antibody titers, assay validation and how they are interpreted are important considerations—but for a fatal disease like rabies, they are of paramount importance. The innate limitations of a one-dimensional laboratory test for rabies antibody measurement, as well as the validation of the method of choice, must be carefully considered in the selection of an assay method and for the interpretation of results that might be construed as a surrogate of protection.

The genus Salmonella has more than 2500 serological variants (serovars), such as Salmonella enterica serovar Typhi and Salmonella Paratyphi A and B, that cause, respectively, typhoid and paratyphoid fevers (enteric fevers), and a large number of non-typhoidal Salmonella (NTS) serovars that cause gastroenteritis in healthy hosts. In young infants, the elderly and immunocompromised hosts, NTS can cause severe, fatal invasive disease. Multiple studies of pediatric patients in sub-Saharan Africa have documented the important role of NTS, in particular Salmonella Typhimurium and Salmonella Enteritidis (and to a lesser degree Salmonella Dublin), as invasive bacterial pathogens. Salmonella spp. are isolated from blood and identified by standard microbiological techniques and the serovar is ascertained by agglutination with commercial antisera. PCR-based typing techniques are becoming increasingly popular in developing countries, in part because high quality typing sera are difficult to obtain and expensive and H serotyping is technically difficult. We have developed a series of polymerase chain reactions (PCRs) to identify Salmonella Typhimurium and variants, Salmonella Enteritidis and Salmonella Dublin. We successfully identified 327 Salmonella isolates using our multiplex PCR. We also designed primers to detect Salmonella Stanleyville, a serovar found in West Africa. Another PCR generally differentiated diphasic Salmonella Typhimurium and monophasic Salmonella Typhimurium variant strains from other closely related strains. The PCRs described here will enable more laboratories in developing countries to serotype NTS that have been isolated from blood.

The Elapidae family is represented in America by three genera of coral snakes: Micruroides, Leptomicrurus and Micrurus, the latter being the most abundant and diversified group. Micrurus bites can cause death by muscle paralysis and respiratory arrest few hours after envenomation. The specific treatment for Micrurus envenomation is the application of heterologous antivenom. The aim of this study was to compare the toxicity of venoms from nine species of coral snakes and analyze the neutralization potential of the Brazilian coral snake antivenom. In vitro assays showed that the majority of the Micrurus venoms are endowed with phospholipase and hyaluronidase and low proteolytic activities. These enzymes are not equally neutralized in all venoms by the therapeutic antivenom. Moreover, in vivo assays showed that some of the Micrurus venoms are extremely lethal, such as the ones from M. altirostris, M. corallinus, M. frontalis, M. lemniscatus and M. spixii. Neutralization tests, performed in vivo, showed that the therapeutic antivenom was able to neutralize better the venoms from M. frontalis, M. corallinus, and M. spixii but not from M. altirostris and M. lemniscatus. Taken together, these results suggest that modifications in the immunization antigenic mixture should occur in order to generate more comprehensive therapeutic antivenom.

Filarial parasites such as Brugia malayi and Onchocerca volvulus are the causative agents of the tropical diseases lymphatic filariasis and onchocerciasis, which infect 150 million people, mainly in Africa and Southeast Asia. Filarial nematodes have a complex life cycle that involves transmission and development within both mammalian and insect hosts. The successful completion of the life cycle includes four molts, two of which are triggered upon transmission from one host to the other, human and mosquito, respectively. Elucidation of the molecular mechanisms involved in the molting processes in filarial nematodes may yield a new set of targets for drug intervention. In insects and other arthropods molting transitions are regulated by the steroid hormone ecdysone that interacts with a specialized hormone receptor composed of two different proteins belonging to the family of nuclear receptors. We have cloned from B. malayi two members of the nuclear receptor family that show many sequence and biochemical properties consistent with the ecdysone receptor of insects. This finding represents the first report of a functional ecdysone receptor homolog in nematodes. We have also established a transgenic hormone induction assay in B. malayi that can be used to discover ecdysone responsive genes and potentially lead to screening assays for active compounds for pharmaceutical development.

Certain sand fly species living inside or on the edge of tropical forests are well known to transmit a protozoan to humans, which in lowland Panama develops into a cutaneous form of leishmaniasis; open, itching sores on the face and extremities requiring aggressive treatment with antimonial compounds. Morphological characters and DNA sequence from mitochondrial and nuclear gene fragments permitted us to identify and then establish historical relationships among 20 common sand fly species occurring in the understory of Barro Colorado Island, a forested preserve in the middle of the Panama Canal. Individuals in three of these sand fly species were found to be 26–43% infected by Leishmania naiffi, a species hitherto known only from the Amazonian region and the Caribbean. We then screened the same 20 sand fly species for the cytoplasmically transmitted bacteria Wolbachia pipientis, finding three infected at high rates, each by a distinct strain. Lutzomyia trapidoi, the most likely transmitter of Leishmania to humans in Panama, was among the Wolbachia-infected species, thus marking it as a possible high-value target for future biocontrol studies using the bacteria either to induce mating incompatabilities or to drive selected genes into the population.

Cutaneous leishmaniasis, a parasitic skin infection transmitted by the bite of a sand fly, can result in chronic skin sores and is estimated to affect more than 1.5 million persons worldwide. While the infection generally heals on its own in months to years, treatment can be expensive and difficult. We compared a heat treatment using the ThermoMed device to an (abbreviated) ten day course of intravenous Pentostam (a pentavalent antimony drug) in a population of U.S. soldiers who acquired their infections in Iraq. We found no statistically significant difference between the two treatments in the healing rate at two months. The heat treatment had less associated toxicity. Heat therapy is a ruggedized, battery operated method that could be adapted to humanitarian situations and less developed health care settings, likely with less cost and side effects than local treatment alternatives.

Plague, caused by Yersinia pestis, is one of the oldest and most dangerous diseases in human history, and has claimed millions of lives in the three major historical pandemics. Although panic caused by the Black Death is fading, the threat of the reemergence of plague pandemics still exists, with the additional potential of misuse in biowarfare or bioterrorism. Rapid on-site detection and identification of the pathogen is of paramount significance for timely implementation of effective countermeasures. TaqMan probe-based real-time PCR assays can give quick and accurate identification; however, the need for cold delivery and storage prevents its potential on-site application. The objective of this study was to develop a stable PCR system for easy delivery and storage under room temperature, which is vital for conventional plague surveillance and for preparedness in public health emergencies. We present a solution to this particular issue, hoping that it is helpful to future applications.

The intermediate host of Schistosoma japonicum in Asia is the snail Oncomelania hupensis, which can be separated phenotypically into ribbed- and smooth-shelled morphotypes. In China, the typical morphotype is ribbed-shelled, with its distribution restricted to mainland China. Smooth-shelled snails with varix are also distributed in China, which are considered to belong to the same subspecies as the ribbed-shelled snails. In this study we investigate the genetic variation among O. hupensis from different geographical origins using combined complete ITS1 and ITS2 regions of nuclear ribosomal DNA. Snails including ribbed-shelled and smooth-shelled (but with varix on the shell) from the lake/marshland region of the middle and lower reaches of the Yangtze River, and smooth-shelled snails from mountainous regions of Sichuan and Yunnan provinces, were genetically distinct with no shared haplotypes detected. Furtheremore, the snails from Sichuan and Yunnan provinces were clustered in separate clades in the phylogenetic tree, and three clades were observed for snails from the middle and lower reaches of the Yangtze River. The population diversity of O. hupensis in China is thus considered large, and evolutionary relationships in the host-parasite system of O. hupensis-S. japonicum may be of interest for further research.

Frequent outbreaks of acute Chagas' disease by food contamination with T. cruzi, characterized by high mortality, have been reported in recent years. In Brazil, oral infection is currently the most important mechanism of T. cruzi transmission. Studies on oral T. cruzi infection in mice have shown that insect-stage metacyclic trypomastigotes invade only the gastric mucosal epithelium and not other areas of mucosal epithelia prior to establishing systemic infection. Here we have shown that metacyclic trypomastigotes bind selectively to gastric mucin, a property also displayed by gp82, a metacyclic stage-specific surface protein implicated in cell adhesion/invasion process. It is also shown that the gastric mucin-binding property of gp82 resides in the central domain of the molecule and that the synthetic peptide p7, based on a gastric mucin-binding sequence of gp82, markedly reduces parasite invasion of cultured human epithelial cells in the presence of gastric mucin. These results, plus the finding that mice that received peptide p7 before oral infection with metacyclic trypomastigotes had fewer parasites replicating in the gastric mucosa and developed lower parasitemias than control mice, lead us to suggest that gp82-mediated interaction with gastric mucin may direct T. cruzi to stomach mucosal epithelium in oral infection.

Intestinal parasitic nematode diseases infect over one billion people and cause significant disease burden in children (growth and cognitive stunting, malnutrition), in pregnant women, and via their dampening of the immune system in infected individuals. In over thirty years, no new classes of anti-roundworm drugs (anthelmintics) for treating humans have been developed. Because of limitations of the current drugs and the threat of parasite resistance, new anthelmintics are needed. The soil bacterium Bacillus thuringiensis (Bt) produces crystal (Cry) proteins that specifically target and kill insects and nematodes and is used around the world as a safe insecticide. Here we test the effects of the Bt Cry protein Cry5B on a chronic, natural intestinal roundworm infection in mice, namely the helminth parasite Heligmosomoides bakeri. We find that a single dose of Cry5B can eliminate 70% of the parasites and can almost completely block the ability of the parasites to produce progeny. Comparisons of Cry5B's efficacy with known anthelmintics suggest its activity is as good as or perhaps even better than those currently used. Furthermore, this protein is rapidly digested by simulated stomach juices, suggesting that protecting it from these juices would reveal a superior anthelmintic.

The four dengue viruses (DENV) represent the most common human arbovirus infections in the world and are currently a challenging problem, particularly in the tropical and subtropical regions of Asia and the Americas. Infection with DENV may produce symptoms of varying severity. While access to care, appropriate interventions, host genetic factors, and previous exposure to DENV are all known to affect the outcome of the infection, it is not entirely understood why some individuals develop more severe disease. It has been hypothesized that the four dengue serotypes differ in disease severity and clinical manifestations. This analysis assessed whether there were significant differences in severity of disease caused by the dengue serotypes in a pediatric population in Thailand. We found significant and non-significant correlations between dengue serotype 2 infection and more severe dengue disease. We also found that individual serotypes varied in disease severity between study years, perhaps supporting the hypothesis that the particular sequences of primary and secondary DENV infections influence disease severity.

Trypanosoma congolense is a parasite responsible for severe disease of African livestock. Its life cycle is complex and divided into two phases, one in the tsetse fly vector and one in the bloodstream of the mammalian host. Molecular tools for gene function analyses in parasitic organisms are essential. Previous studies described the possibility of completing the entire T. congolense life cycle in vitro. However, the model showed major flaws including the absence of stable long-term culture of the infectious bloodstream forms, a laborious time-consuming period to perform the cycle and a lack of genetic tools. We therefore aimed to develop a standardized model convenient for genetic engineering. We succeeded in producing long-term cultures of all the developmental stages on long-term, to define all the differentiation steps and to finally complete the whole cycle in vitro. This improved model offers the opportunity to conduct phenotype analyses of genetically modified strains throughout the in vitro cycle and also during experimental infections.

Blood-sucking bugs of the genus Rhodnius are major vectors of Chagas disease. Control and surveillance of Chagas disease transmission critically depend on ascertaining whether households and nearby ecotopes (such as palm trees) are infested by these vectors. However, no bug detection technique works perfectly. Because more sensitive methods are more costly, vector searches face a trade-off between technical prowess and sample size. We compromise by using relatively inexpensive sampling techniques that can be applied multiple times to a large number of palms. With these replicated results, we estimate the probability of failing to detect bugs in a palm that is actually infested. We incorporate this information into our analyses to derive an unbiased estimate of palm infestation, and find it to be about 50% – twice the observed proportion of infested palms. We are then able to model the effects of regional, landscape, and local environmental variables on palm infestation. Individual palm attributes contribute overwhelmingly more than landscape or regional covariates to explaining infestation, suggesting that palm tree management can help mitigate risk locally. Our results illustrate how explicitly accounting for vector, pathogen, or host detection failures can substantially improve epidemiological parameter estimation when perfect detection techniques are unavailable.

Macrophage migration inhibitory factor (MIF) has emerged as a pivotal mediator of innate immunity and has been shown to be an important effector molecule in severe sepsis. Melioidosis, caused by Burkholderia pseudomallei, is an important cause of community-acquired sepsis in Southeast Asia. Here an immunostain for MIF is shown in the lungs of mice infected with B. pseudomallei. Positive immunostaining for MIF can be seen in the epithelial submucosa, bronchial epithelial cells, and inflammatory cells, most notably of alveolar macrophages (magnification x 10). MIF concentrations are markedly elevated during melioidosis and correlate with poor outcome. During experimental melioidosis MIF impairs antibacterial defense. (See article by Wiersinga et al., doi:10.1371/journal.pntd.0000605).

Image Credit: Sandrine Florquin (Department of Pathology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands).

Once infected with dengue virus, a female Aedes aegypti mosquito must survive longer than twelve days before it can transmit the virus to an uninfected person. New dengue control strategies therefore aim to circumvent dengue transmission using entomopathogenic microorganisms that shorten mosquito lifespan. Accurate methods to determine the age of individual mosquitoes are required for these and other mosquito control interventions. We have previously shown that mosquito age can be predicted from the transcription of specific genes. Here we demonstrate that this can be achieved under natural conditions when mosquitoes are uncaged and free to engage in natural behavior. To do this, we produced “free-range” female mosquitoes by releasing 8007 mosquitoes at an isolated location and recapturing the females of known ages. We developed an age prediction model from gene transcription measures of mosquitoes maintained in small “sentinel cages” maintained onsite. We then used this model to predict the ages of the free-range mosquitoes, based on their own transcription measures. Age predictions were robust to physiological changes associated with blood feeding and egg development. We show that the technique could be applied to identify a 50% reduction in mosquito population survival that is expected from infection with entomopathogenic Wolbachia bacteria.

Symptoms and signs of leptospirosis are non-specific. A number of diagnostic tests for leptospirosis are available. We compared the cost-benefit of 5 management strategies: 1) no patients tested or given antibiotic treatment; 2) all patients given empirical doxycycline treatment; patients given doxycycline when a patient is tested positive for leptospirosis using: 3) lateral flow; 4) MCAT; 5) latex test. Outcomes were measured in duration of fever which is then converted to productivity losses to capture the full economic costs. Empirical doxycycline treatment was found to be the most efficient strategy, being both the least costly alternative and the one that resulted in the lowest average duration of fever. The significantly higher relative cost of using a diagnostic test as compared with presumptive treatment, and the limited sensitivity of all the diagnostic tests implied that only the latex test could be considered cost-effective when compared with the no-antibiotic-treatment option, and that all three tests were still inferior to empirical treatment.

Leptospirosis is a bacterial disease commonly transmitted from animals to humans. The more than 200 types of spiral-shaped bacteria (spirochetes) in the genus Leptospira are classified as pathogenic, intermediately pathogenic, or saprophytic (meaning not causing infection in any mammal) based on their ability to cause disease and on genetic information. Unique among the spirochetes that infect humans, Leptospira live both in the environment (in surface waters and moist soils), and in mammals, where they cause chronic infection by colonizing kidney tubules. Infected animals are the source of human infection, but humans have not been systematically studied as chronic Leptospira carriers. In our study, we found that more than 5% of people (in fact, only women) in a rural Amazonian village, without clinical evidence of infection by Leptospira, were chronically colonized by the bacteria. Chronic infection was not associated with a detectable immune response against the spirochete. Pathogenic and intermediately pathogenic Leptospira caused asymptomatic, chronic kidney infections. Future work is needed to determine whether such chronic infection can lead to human-to-human transmission of leptospirosis, and whether subtle measures of kidney disease are associated with asymptomatic, long-term leptospiral infection.

Cystic Echinococcosis (CE) is a near-cosmopolitan parasitic zoonosis that causes economic losses in many regions of the world. This parasitic infection can be regarded as an emerging or re-emerging disease causing considerable losses in livestock production. CE is produced by the larval cystic stage (hydatid) of the dog parasite Echinococcus granulosus. After infective eggs are ingested, cysts develop mainly in lungs and liver of humans and animals (sheep, cattle, pigs, horses, etc). Infected people may require surgery and/or Albendazole-based chemotherapy. In this study, we evaluated the effects of Oxfendazole alone (an antiparasitic drug used in animals), Oxfendazole plus Praziquantel, and Albendazole plus Praziquantel against hydatid cysts in sheep over 4 to 6 weeks of treatment. All of the treatments in this study were efficacious in killing the larval stages and, therefore, in minimizing the risk of a dog acquiring new infections (taenias). These treatment schemes can be added to control measures in animals and eventually could be used for the treatment of human infection. Further investigations on different schedules of monotherapy or combined chemotherapy are needed, as well as studies to evaluate the safety and efficacy of Oxfendazole in humans.

Elimination of canine rabies has been achieved in some parts of the world, but the disease still kills many thousands of people each year in Africa. Here we counter common arguments given for the lack of effective canine rabies control in Africa presenting detailed data from a range of settings. We conclude that (1) rabies substantially affects public and animal health sectors, hence regional and national priorities for control ought to be higher, (2) for practical purposes domestic dogs are the sole maintenance hosts and main source of infection for humans throughout most of Africa and Asia and sufficient levels of vaccination coverage in domestic dog populations should lead to elimination of canine rabies in most areas, (3) the vast majority of domestic dog populations across sub-Saharan Africa are accessible for vaccination with community sensitization being of paramount importance for the success of these programs, (4) improved local capacity in rabies surveillance and diagnostics will help evaluate the impact of control and elimination efforts, and (5) sustainable resources for effective dog vaccination campaigns are likely to be available through the development of intersectoral financing schemes involving both medical and veterinary sectors.