Human Disease-causing Pathogens Research Articles

A next generation sequencing assay combining Ixodes species identification with pathogen detection to support tick surveillance efforts in the United States

The burden of tick-borne diseases continues to increase in the United States. Tick surveillance has been implemented to monitor changes in the distribution and prevalence of human disease-causing pathogens in ticks that frequently bite humans. Such efforts require accurate identification of ticks to species and highly sensitive and specific assays that can detect and differentiate pathogens from genetically similar microbes in ticks that have not been demonstrated to be pathogenic in humans. We describe a modification to a next generation sequencing pathogen detection assay that includes a target that accurately identifies Ixodes ticks to species. We show that the replacement of internal control primers used to ensure assay performance with primers that also act as an internal control and can additionally differentiate tick species, retains high sensitivity and specificity, improves efficiency, and reduces costs by eliminating the need to run separate assays to screen for pathogens and for tick identification.

Roles of ADP-Ribosylation during Infection Establishment by Trypanosomatidae Parasites.

ADP-ribosylation is a reversible post-translational protein modification, which is evolutionarily conserved in prokaryotic and eukaryotic organisms. It governs critical cellular functions, including, but not limited to cellular proliferation, differentiation, RNA translation, and genomic repair. The addition of one or multiple ADP-ribose moieties can be catalysed by poly(ADP-ribose) polymerase (PARP) enzymes, while in eukaryotic organisms, ADP-ribosylation can be reversed through the action of specific enzymes capable of ADP-ribose signalling regulation. In several lower eukaryotic organisms, including Trypanosomatidae parasites, ADP-ribosylation is thought to be important for infection establishment. Trypanosomatidae encompasses several human disease-causing pathogens, including Trypanosoma cruzi, T. brucei, and the Leishmania genus. These parasites are the etiological agents of Chagas disease, African trypanosomiasis (sleeping sickness), and leishmaniasis, respectively. Currently, licenced medications for these infections are outdated and often result in harmful side effects, and can be inaccessible to those carrying infections, due to them being classified as neglected tropical diseases (NTDs), meaning that many infected individuals will belong to already marginalised communities in countries already facing socioeconomic challenges. Consequently, funding to develop novel therapeutics for these infections is overlooked. As such, understanding the molecular mechanisms of infection, and how ADP-ribosylation facilitates infection establishment by these organisms may allow the identification of potential molecular interventions that would disrupt infection. In contrast to the complex ADP-ribosylation pathways in eukaryotes, the process of Trypanosomatidae is more linear, with the parasites only expressing one PARP enzyme, compared to the, at least, 17 genes that encode human PARP enzymes. If this simplified pathway can be understood and exploited, it may reveal new avenues for combatting Trypanosomatidae infection. This review will focus on the current state of knowledge on the importance of ADP-ribosylation in Trypanosomatidae during infection establishment in human hosts, and the potential therapeutic options that disrupting ADP-ribosylation may offer to combat Trypanosomatidae.

Ontogenetic Changes in Nutrients and Stoichiometry in the Invasive Mosquito, Aedes albopictus (Diptera: Culicidae).

A variety of physiological, morphological, and behavioral changes occur throughout the life cycle of mosquitoes, which can be correlated with a shift from the aquatic to terrestrial environment. Aedes albopictus Skuse is an abundant invasive species from Asia that was introduced into the Americas in the 1980's and is responsible for transmitting several important human disease-causing pathogens. How physiological and anatomical changes within each instar and throughout the developmental stages are related to changes in carbon (C) and nitrogen (N) levels are an unexplored area of mosquito ecology. We hypothesized that these changes as well as stoichiometry (C:N) would vary with instar stage and larval diet. Cohorts of larvae were grown in three different diets: animal only (crickets), plant only (red maple leaves), and a mixture containing both types. Larval instars (1st-4th), pupae, and adults were raised in each diet and were separately analyzed for nutrient content (%C, %N) and stoichiometry (C:N). Significant changes in nutrient values occurred across the life cycle, with C:N values being lower in early instars versus adults or pupae, especially in animal only or mixed diets; few differences were detected in %C or %N across ontogeny. This knowledge may lead to a better understanding of mosquito ecology and pathogen transmission.

In vivo pair correlation microscopy reveals dengue virus capsid protein nucleocytoplasmic bidirectional movement in mammalian infected cells

Flaviviruses are major human disease-causing pathogens, including dengue virus (DENV), Zika virus, yellow fever virus and others. DENV infects hundreds of millions of people per year around the world, causing a tremendous social and economic burden. DENV capsid (C) protein plays an essential role during genome encapsidation and viral particle formation. It has been previously shown that DENV C enters the nucleus in infected cells. However, whether DENV C protein exhibits nuclear export remains unclear. By spatially cross-correlating different regions of the cell, we investigated DENV C movement across the nuclear envelope during the infection cycle. We observed that transport takes place in both directions and with similar translocation times (in the ms time scale) suggesting a bidirectional movement of both C protein import and export.Furthermore, from the pair cross-correlation functions in cytoplasmic or nuclear regions we found two populations of C molecules in each compartment with fast and slow mobilities. While in the cytoplasm the correlation times were in the 2–6 and 40–110 ms range for the fast and slow mobility populations respectively, in the cell nucleus they were 1–10 and 25–140 ms range, respectively. The fast mobility of DENV C in cytoplasmic and nuclear regions agreed with the diffusion coefficients from Brownian motion previously reported from correlation analysis. These studies provide the first evidence of DENV C shuttling from and to the nucleus in infected cells, opening new venues for antiviral interventions.

Exploring the Niche of Rickettsia montanensis (Rickettsiales: Rickettsiaceae) Infection of the American Dog Tick (Acari: Ixodidae), Using Multiple Species Distribution Model Approaches.

The American dog tick, Dermacentor variabilis (Say) (Acari: Ixodidae), is a vector for several human disease-causing pathogens such as tularemia, Rocky Mountain spotted fever, and the understudied spotted fever group rickettsiae (SFGR) infection caused by Rickettsia montanensis. It is important for public health planning and intervention to understand the distribution of this tick and pathogen encounter risk. Risk is often described in terms of vector distribution, but greatest risk may be concentrated where more vectors are positive for a given pathogen. When assessing species distributions, the choice of modeling framework and spatial layers used to make predictions are important. We first updated the modeled distribution of D. variabilis and R. montanensis using maximum entropy (MaxEnt), refining bioclimatic data inputs, and including soil variables. We then compared geospatial predictions from five species distribution modeling frameworks. In contrast to previous work, we additionally assessed whether the R. montanensis positive D. variabilis distribution is nested within a larger overall D. variabilis distribution, representing a fitness cost hypothesis. We found that 1) adding soil layers improved the accuracy of the MaxEnt model; 2) the predicted ‘infected niche’ was smaller than the overall predicted niche across all models; and 3) each model predicted different sizes of suitable niche, at different levels of probability. Importantly, the models were not directly comparable in output style, which could create confusion in interpretation when developing planning tools. The random forest (RF) model had the best measured validity and fit, suggesting it may be most appropriate to these data.

Halictine-2 antimicrobial peptide shows promising anti-parasitic activity against Leishmania spp.

The protozoan parasite Leishmania spp. causes leishmaniases, a group of diseases creating serious health problems in many parts of the world with significant resistance to existing drugs. Insect derived antimicrobial peptides are promising alternatives to conventional drugs against several human disease-causing pathogens because they do not generate resistance. Halictine-2, a novel antimicrobial peptide from the venom of eusocial honeybee, Halictus sexcinctus showed significant anti-leishmanial activity in vitro, towards two life forms of the dimorphic parasite, the free-swimming infective metacyclic promastigotes and the intracellular amastigotes responsible for the systemic infection. The anti-leishmanial activity of the native peptide (P5S) was significantly enhanced by serine to threonine substitution at position 5 (P5T). The peptide showed a propensity to form α-helices after substitution at position-5, conferring amphipathicity. Distinct pores observed on the promastigote membrane after P5T exposure suggested a mechanism of disruption of cellular integrity. Biochemical alterations in the promastigotes after P5T exposure included generation of increased oxygen radicals with mitochondrial Ca2+ release, loss of mitochondrial membrane potential, reduction in total ATP content and increased mitochondrial mass, resulting in quick bioenergetic and chemiosmotic collapse leading to cell death characterized by DNA fragmentation. P5T was able to reduce intracellular amastigote burden in an in vitro model of Leishmania infection but did not alter the proinflammatory cytokines like TNF-α and IL-6. The ability of the P5T peptide to kill the Leishmania parasite with negligible haemolytic activity towards mouse macrophages and human erythrocytes respectively, demonstrates its potential to be considered as a future antileishmanial drug candidate.

Current Flavivirus Research Important for Vaccine Development

The Flaviviridae family of RNA viruses includes numerous human disease-causing pathogens that largely are increasing in prevalence due to continual climate change, rising population sizes and improved ease of global travel [...].

Surveillance of Culicine Mosquitoes in Six Villages of Taita-Taveta County, Kenya, With Host Determinations From Blood-Fed Females.

Culicine mosquitoes are vectors of human disease-causing pathogens like filarial worms and several arthropod-borne viruses (arboviruses). Currently, there has been an increase in emerging and re-emerging vector-borne diseases along coastal Kenya, which has been of major concern in public health. This study aimed at determining culicine mosquito species abundance, diversity and their host feeding preferences in Taita-Taveta County, Coastal Kenya. Entomological sampling was done during the long-wet season (March and May) and long dry season (June to October) 2016-2018. Mosquito sampling was done using CDC light traps and Backpack aspiration for indoor and outdoor environments. All culicine mosquitoes collected were identified morphologically and categorized according to their physiological status. Blood fed culicine mosquitoes were tested for bloodmeal sources using ELISA. In total, 3,278 culicine mosquitoes were collected, of which 738 (22.5 %) were found indoors and 2,540, (77.5 %) outdoors. The mosquitoes consisted of 18 species belonging to four genera: Aedes (7), Culex (8), Mansonia (2), and Coquillettidia (1). Overall, there was high mosquito species diversity (H) in outdoors (H = 2.4339) than in indoors (H = 2.2523), whereas even distribution (EH) was higher in indoors (EH = 0.9064) than outdoors (EH = 0.8266). Majorly the bloodmeals identified were from multiple host sources with (51.6%), single hosts (41.3%), and unidentified (7.2%). This study has demonstrated a high diversity of culicine mosquitoes with relaxed feeding tendencies. These mosquitoes are contributing to mosquito biting nuisance and the likelihood of exposure of populations to diseases of public health.

A seven-legged tick: Report of a morphological anomaly in Ixodes scapularis (Acari: Ixodidae) biting a human host from the Northeastern United States

Cases of morphological anomalies in the blacklegged tick, Ixodes scapularis (Acari: Ixodidae), have recently been reported from the Northeastern and upper Midwestern United States, potentially complicating identification of this important vector of human disease-causing pathogens. We hereby report a case of a morphological anomaly in I. scapularis, biting a human host residing in Norwich, Connecticut. Using a dichotomous morphological key, high-resolution and scanning electron microscopy images, as well as DNA sequencing, the tick was identified as an adult female I. scapularis with three legs on the left side of the abdomen versus four on the right side, which we believe is the first case of ectromely in an adult I. scapularis. Using diagnostic genes in polymerase chain reaction, the specimen tested positive for Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum, the causative agents for Lyme disease and anaplasmosis, respectively, and also showed evidence of a rickettsial endosymbiont. Here we discuss recent reports of morphological anomalies in I. scapularis, and emphasize the significance of additional studies of teratology in this important tick species and its potential implications.

Insecticidal activity of Artemisia sieberi, Eucalyptus camaldulensis, Thymus persicus and Eruca sativa oils against German cockroach, Blattella germanica (L.)

The German cockroach, Blattella germanica (L.) (Blattodea: Blattellidae), has been recognized as a serious health problem because of its potential to harbor and transmit human disease-causing pathogens. The development of new and environmentally friendly control agents is essential as alternative tool to conventional chemical insecticides. The aim of the current study was to determine the fumigant and contact toxicity of essential oils (EOs), Artemisia sieberi Besser, Eucalyptus camaldulensis Dehn. and Thymus persicus (Ronniger ex Rech. f.) and cold press oil of Eruca sativa (Miller) (ESO) against first instar nymphs and adults. According to results, the LC50 values of EOs were between 15.01 and 28.80 μl/l air for fumigant toxicity, 2202.8 and 5600.2 mg/l for dipping toxicity and 9484.2 and 34,421.1 mg/l for applied surface toxicity. Fumigant activity of the oils were significantly more toxic than other methods. In general, the toxicity of A. sieberi was higher than other EOs. Also, the first instar nymphs were more susceptible than adults. Additionally, the results showed that the EOs were significantly more toxic than ESO in both fumigant and dipping methods. In addition to contact effects, the fumigant activity of ESO was proved, although the toxicity was lower than the EOs. Hence, it is important to consider the production cost benefit of ESO in comparison with the EOs. So, the ESO could be considered as a good candidate in control of the cockroach but in enclosed spaces such as inaccessible cockroach hiding places, the EO of A. sieberi can be useful.

Wolbachia detection in insects through LAMP: loop mediated isothermal amplification.

BackgroundThe bacterium Wolbachia is a promising agent for the biological control of vector-borne diseases as some strains have the ability to block the transmission of key human disease-causing pathogens. Fast, accurate and inexpensive methods of differentiating between infected and uninfected insects will be of critical importance as field-based trials of Wolbachia-based bio-control become increasingly common.FindingsWe have developed a specific and sensitive method of detecting Wolbachia based on the isothermal DNA amplification. This technique can be performed in an ordinary heat block without the need for gel-based visualisation, and is effective for a wide variety of insect hosts.ConclusionHere we present the development of a rapid, highly sensitive and inexpensive method to detect Wolbachia in a variety of insect hosts, including key mosquito disease vectors.

Design and one-pot synthesis of hybrid thiazolidin-4-one-1,3,5-triazines as potent antibacterial agents against human disease-causing pathogens

An efficient and general one-pot reaction to a novel series of hybrid thiazolidine-4-one-1,3,5-triazine derivatives was developed. The easy work-up of the products, rapid reaction, and mild conditions are notable features of this protocol. These molecules were found to exhibit potent activity against a panel of Gram-positive and Gram-negative micro-organisms.

Mosquito species abundance and diversity in Malindi, Kenya and their potential implication in pathogen transmission

Mosquitoes (Diptera: Culicidae) are important vectors of human disease-causing pathogens. Mosquitoes are found both in rural and urban areas. Deteriorating infrastructure, poor access to health, water and sanitation services, increasing population density, and widespread poverty contribute to conditions that modify the environment, which directly influences the risk of disease within the urban and peri-urban ecosystem. The objective of this study was to evaluate the mosquito vector abundance and diversity in urban, peri-urban, and rural strata in Malindi along the Kenya coast. The study was conducted in the coastal district of Malindi between January and December 2005. Three strata were selected which were described as urban, peri-urban, and rural. Sampling was done during the wet and dry seasons. Sampling in the wet season was done in the months of April and June to cover the long rainy season and in November and December to cover the short rainy season, while the dry season was between January and March and September and October. Adult mosquito collection was done using Pyrethrum Spray Collection (PSC) and Centers for Disease Control and Prevention (CDC) light traps inside houses and specimens were identified morphologically. In the three strata (urban, peri-urban, and rural), 78.5% of the total mosquito (n = 7,775) were collected using PSC while 18.1% (n = 1,795) were collected using the CDC light traps. Using oviposition traps, mosquito eggs were collected and reared in the insectary which yielded 329 adults of which 83.8% (n = 276) were Aedes aegypti and 16.2% (n = 53) were Culex quinquefasciatus. The mosquito distribution in the three sites varied significantly in each collection site. Anopheles gambiae, Anopheles funestus and Anopheles coustani were predominant in the rural stratum while C. quinquefasciatus was mostly found in urban and peri-urban strata. However, using PSC and CDC light trap collection techniques, A. aegypti was only found in urban strata. In the three strata, mosquitoes were mainly found in high numbers during the wet season. Further, A. gambiae, C. quinquefasciatus, and A. aegypti mosquitoes were found occurring together inside the houses. This in turn exposes the inhabitants to an array of mosquito-borne diseases including malaria, bancroftian filariasis, and arboviruses (dengue fever, Yellow fever, Rift Valley fever, Chikungunya fever, and West Nile Virus). In conclusion, our findings provide useful information for the design of integrated mosquito and disease control programs in East African environments.

Human Pathogens and Fresh Produce: Prevention and Damage Control: Introduction to the Colloquium

A few years ago, food safety in fresh produce was discussed primarily in terms of pesticide residues. The assumption was that human pathogens just weren’t found on fresh fruits and vegetables, at least not in the United States and Canada, where potable water and chemical fertilizers are used for most production. Suddenly, the produce industry found itself caught up in a new definition of food safety, that of the human disease-causing pathogens, that have plagued the dairy and meat industries. The assumption has been that the pH of fruits and vegetables generally is too low to support the growth of human disease-causing pathogens (Beauchat, 1996). The outbreaks of Escherichia coli O:157:H7 in nonpasteurized apple juice and cider, and the growth of Salmonella on tomatoes (Lycopersicon esculentum Mill.), indicate the need for more vigilant attention to production and handling practices. While the incidence of food pathogen outbreaks in fresh produce is still very low compared with that in other foods, the media attention focused on each new event sparked a new wave of consumer fear about the safety of agricultural commodities. Where did these organisms come from? Several theories have been advanced. One is that microbes have evolved to more pathogenic forms or to forms that can now survive under conditions that previously checked growth, like refrigeration and acidic substrates (Thayer and Reykowski, 1999). Changes in production practices and increased numbers of people handling the same produce may be other factors (Thayer and Reykowski, 1999). The United States and Canada supposedly have the safest food supply in the world. Certainly, this remains true, but the amount of negative publicity surrounding an outbreak can be devastating for both the individual producer and the industry as a whole. The intent of this colloquium is to better understand how these outbreaks occur, how illnesses are documented and traced back to a source, and how steps can be taken at the grower level to minimize the potential for contamination of fresh produce by pathogens causing disease in humans.