Bacterial and parasite co-infection in Mexican golden trout (Oncorhynchus chrysogaster) by Aeromonas bestiarum, Aeromonas sobria, Plesiomonas shigelloides and Ichthyobodo necator.

BACKGROUND: Bacterial infections are responsible of high economic losses in aquaculture. Mexican golden trout (Oncorhynchus chrysogaster) is a threatened native trout species that has been introduced in aquaculture both for species conservation and breeding for production and for which no studies of bacterial infections have been reported. CASE PRESENTATION: Fish from juvenile stages of Mexican golden trout showed an infectious outbreak in a farm in co-culture with rainbow trout (Oncorhynchus mykiss), showing external puntiform red lesions around the mouth and caudal pedunculus resembling furuncles by Aeromonas spp. and causing an accumulated mortality of 91%. Isolation and molecular identification of bacteria from lesions and internal organs showed the presence of Aeromonas bestiarum, Aeromonas sobria, Plesiomonas shigelloides and Ichthyobodo necator isolated from a single individual. All bacterial isolates were resistant to amoxicillin-clavulanic acid and cefazoline. P. shigelloides was resistant to third generation ß-lactamics. CONCLUSIONS: This is the first report of coinfection by Aeromonas bestiarum, Aeromonas sobria, Plesiomonas shigelloides and Ichthyobodo necator in an individual of Mexican golden trout in co-culture with rainbow trout. Resistance to ß-lactams suggests the acquisition of genetic determinants from water contamination by human- or livestock-associated activities.

Differential drivers of intraspecific and interspecific competition during malaria-helminth co-infection.

Various host and parasite factors interact to determine the outcome of infection. We investigated the effects of two factors on the within-host dynamics of malaria in mice: initial infectious dose and co-infection with a helminth that limits the availability of red blood cells (RBCs). Using a statistical, time-series approach to model the within-host 'epidemiology' of malaria, we found that increasing initial dose reduced the time to peak cell-to-cell parasite propagation, but also reduced its magnitude, while helminth co-infection delayed peak cell-to-cell propagation, except at the highest malaria doses. Using a mechanistic model of within-host infection dynamics, we identified dose-dependence in parameters describing host responses to malaria infection and uncovered a plausible explanation of the observed differences in single vs co-infections. Specifically, in co-infections, our model predicted a higher background death rate of RBCs. However, at the highest dose, when intraspecific competition between malaria parasites would be highest, these effects of co-infection were not observed. Such interactions between initial dose and co-infection, although difficult to predict a priori, are key to understanding variation in the severity of disease experienced by hosts and could inform studies of malaria transmission dynamics in nature, where co-infection and low doses are the norm.

Allele-Specific Detection Methods for QoI Fungicide-Resistant Erysiphe necator in Vineyards.

Grapevine powdery mildew (GPM), caused by the fungus Erysiphe necator, is a constant threat to worldwide production of grape berries, requiring repeated use of fungicides for management. The frequent fungicide applications have resulted in resistance to commonly used quinone outside inhibitor (QoI) fungicides and the resistance is associated with single-nucleotide polymorphisms (SNPs) in the mitochondrial cytochrome b gene (cytb). In this study, we attempted to detect the most common SNP causing a glycine to alanine substitution at amino acid position 143 (i.e., G143A) in the cytb protein, to track this resistance using allele-specific TaqMan probe and digital-droplet PCR-based assays. Specificity and sensitivity of these assays showed that these two assays could discriminate SNPs and were effective on mixed samples. These diagnostic assays were implemented to survey E. necator samples collected from leaf and air samples from California and Oregon grape-growing regions. Sequencing of PCR amplicons and phenotyping of isolates also revealed that these assays accurately detected each allele (100% agreement), and there was an absolute agreement between the presence or absence of the G143A mutation and resistance to QoIs in the E. necator sampled. These results indicate that the developed diagnostic tools will help growers make informed decisions about fungicide selections and applications which, in turn, will facilitate GPM disease management and improve grape production systems.

Genetic diversity of primate strongylid nematodes: Do sympatric nonhuman primates and humans share their strongylid worms?

The close phylogenetic relationship between humans and nonhuman primates (NHPs) can result in a high potential for pathogen exchange. In recent decades, NHP and human interactions have become more frequent due to increasing habitat encroachment and ecotourism. Strongylid communities, which include members of several genera, are typically found in NHPs. Using optimized high-throughput sequencing for strain-level identification of primate strongylids, we studied the structure of strongylid communities in NHPs and humans co-habiting a tropical forest ecosystem in the Central African Republic. General taxonomic assignment of 85 ITS-2 haplotypes indicated that the studied primates harbour at least nine genera of strongylid nematodes, with Oesophagostomum and Necator being the most prevalent. We detected both host-specific and shared strongylid haplotypes. Skin-penetrating Necator gorillaehaplotypes were shared between humans and gorillas but Necator americanus were much more restricted to humans. Strongylid communities of local hunter-gatherers employed as trackers were more similar to those of gorillas compared to their relatives, who spent more time in villages. This was due to lower abundance of human-origin N. americanus in both gorillas and trackers. Habituated gorillas or those under habituation did not show larger overlap of strongylids with humans compared to unhabituated. We concluded that the occurrence of the human-specific strongylids in gorillas does not increase with direct contact between gorillas and humans due to the habituation. Overall, our results indicate that the degree of habitat sharing between hosts, together with mode of parasite transmission, are important factors for parasite spillover among primates.

Efficacy of single-dose 500 mg mebendazole in soil-transmitted helminth infections: a review.

Soil-transmitted helminthiasis (STH) is caused by Ascaris lumbricoides (roundworm), Trichuris trichiura (whipworm), and Ancylostoma duodenale and Necator americanus (hookworms). Mebendazole is one of the recommended preventive chemotherapy agents for STH. This review summarizes the efficacy data from 29 studies with single-dose 500 mg mebendazole in STH treatment and compares the results with those of a recently conducted phase 3 study of a 500 mg mebendazole chewable tablet against A. lumbricoides and T. trichiura infections. Studies that reported efficacy results against at least one STH infection were selected from the literature and efficacy data by each STH type were abstracted and pooled. Single-dose 500 mg mebendazole treatment resulted in a cure rate of 92.6% (range: 72.5-100%) for A. lumbricoides, 27.6% (range: 8.4-100%) for T. trichiura and 25.5% (range: 2.9-91.1%) for hookworms. Egg reduction rate for A. lumbricoides was 97.9% (range: 89.8-100%), for T. trichiura it was 72.9% (range: 31.6-93.0%) and for hookworms it was 72.0% (range: -6.5% (denoting an increase in egg count) to 98.3%). Similar results were observed in the studies that were placebo-controlled. In the phase 3 study, the cure rate and egg reduction rate reported was 83.7% and 97.9%, respectively, for A. lumbricoides and 33.9% and 59.7%, respectively, for T. trichiura. In conclusion, single-dose 500 mg mebendazole showed a high cure rate against A. lumbricoides and a substantial reduction in faecal egg count for all STH types. These results are consistent with the recently conducted phase 3 study of a new 500 mg chewable mebendazole tablet.

No impact of strongylid infections on the detection of Plasmodium spp. in faeces of western lowland gorillas and eastern chimpanzees.

BACKGROUND: Although a high genetic diversity of Plasmodium spp. circulating in great apes has been revealed recently due to non-invasive methods enabling detection in faecal samples, little is known about the actual mechanisms underlying the presence of Plasmodium DNA in faeces. Great apes are commonly infected by strongylid nematodes, including hookworms, which cause intestinal bleeding. The impact of strongylid infections on the detection of Plasmodium DNA in faeces was assessed in wild, western, lowland gorillas from Dzanga Sangha Protected Areas, Central African Republic and eastern chimpanzees from Kalinzu Forest Reserve, Uganda. METHODS: Fifty-one faecal samples from 22 habituated gorillas and 74 samples from 15 habituated chimpanzees were analysed using Cytochrome-b PCR assay and coprological methods. RESULTS: Overall, 26.4% of the analysed samples were positive for both Plasmodium spp. and strongylids. However, the results showed no significant impact of intensity of infections of strongylids on detection of Plasmodium DNA in gorilla and chimpanzee faeces. CONCLUSION: Bleeding caused by strongylid nematode Necator spp. cannot explain the presence of Plasmodium DNA in ape faeces.

Restoration of hookworm egg development after prolonged storage in stool suspension.

Hookworm infection is still prevalent in southern Thailand despite control measures. Hookworm eggs submerged for an extended period under water from rainfall or in latrines may not survive, but they may recover their ability to develop into infective larvae when exposed to atmospheric air. This study examined the survival of the hookworm eggs in stool suspension and the restoration of development capability after prolonged storage. In stool mass, eggs developed normally and yielded infective filariform larvae (FL) in 7 days. On the contrary, in 1:10 stool suspension, hookworm eggs were found to remain at the 4-8 cell stage; degenerated eggs were observed after 15 days of storage, and the number of degenerated eggs reached 80 % on day 30. Aeration of the suspension, or transferring to a Petri dish or agar plate, restored the capacity of eggs stored for up to 15 days to develop into FL; thereafter, the capacity declined sharply. Retardation of egg development under water or in stool suspension may be due to a lack of atmospheric air. Use of "night soil" from latrines as fertilizer may be one factor in maintaining hookworm transmission, as worm eggs can undergo normal development upon exposure to atmospheric air.

The production of Necator americanus larvae for use in experimental human infection.

BACKGROUND: Although there is unprecedented interest in experimental human hookworm infection, details of hookworm manufacture and characterisation have been sparsely reported. In this report, we detail the production and characterisation of Necator americanus larvae for use in a recently published clinical trial. METHODS: Faeces was obtained from an experimentally infected donor. Faecal hookworm DNA was determined by quantitative PCR. Paired samples were incubated in either sterile water or sterile water mixed with antimicrobials (amphotericin and gentamicin). Coproculture was performed by modified Harada-Mori method. The harvested larvae were then processed in either sterile water or antiseptic solution. Larval yield was then calculated (larvae per gram), larval viability was determined by thermally induced motility assay and microbial burden was determined at the day of harvest, at 48 h and at 7 days. RESULTS: Twenty-eight faecal cultures were performed over 16 months. The faecal hookworm DNA content was variable over this time. There was no association of larval yield with faecal hookworm DNA content. Pre-treatment of faeces with antimicrobials did not influence larval yield. Larval motility was 85.3% (95% CI 79.3-91.3%). Incubation of larvae in antiseptics did not reduce viability at 14 days with a marginal mean of 68.6% (95% CI 59.1-78.1%) washed in water vs. 63.3% (95% CI 53.8 - 72.9%) when incubated in betadine (p = 0.38). Larvae washed in sterile water did not meet microbial bioburden criteria. Incubation in antiseptic resulted in acceptable microbial bioburden at 48 h but not at 7 days. Although the addition of gentamicin did reduce the microbial bio-burden acceptable levels, it was found to significantly lower larval motility at 7 days compared to incubation in sterile water and motility at 7 days 37.8% (95% CI 4.7-70.9%) vs. 67.3% (95% CI 35.2-99.3%, p < 0.001), respectively. CONCLUSIONS: Despite standardised culture methodologies and the use of a single donor, larval yield varied considerably between batches and had no association with faecal hookworm DNA. Larval viability decreases over time and the age of larvae at time of use are likely to be important. Microbial bioburden maybe temporarily reduced by incubation in antiseptics and has little effect on viability. Incubation of larvae in gentamicin is effective at reducing microbial bioburden but is deleterious to larval viability.

Detection of DNA of filariae closely related to Mansonella perstans in faecal samples from wild non-human primates from Cameroon and Gabon.

BACKGROUND: The Onchocercidae is a family of filarial nematodes with several species of medical or veterinary importance. Microfilariae are found in the blood and/or the dermis and are usually diagnosed in humans by microscopy examination of a blood sample or skin biopsy. The main objectives of this study were to evaluate whether filariae DNA can be detected in faecal samples of wild non-human primates (NHPs), whether the detected parasites were closely related to those infecting humans and whether filarial DNA detection in faeces is associated with co-infections with nematodes (Oesophagostumum sp. and Necator sp.) known to cause blood loss while feeding on the host intestinal mucosa. METHODS: A total of 315 faecal samples from 6 species of NHPs from Cameroon and Gabon were analysed. PCRs targeted DNA fragments of cox1 and 12S rDNA genes, to detect the presence of filariae, and the internal transcribed spacer 2 (ITS2), to detect the presence of Oesophagostomum sp. and Necator sp. infections. RESULTS: Among the 315 samples analysed, 121 produced sequences with > 90% homology with Onchocercidae reference sequences. However, 63% of the 12S rDNA and 78% of the cox1 gene sequences were exploitable for phylogenetic analyses and the amplification of the 12S rDNA gene showed less discriminating power than the amplification of the cox1 fragment. Phylogenetic analyses showed that the cox1 sequences obtained from five chimpanzee DNA faecal samples from Gabon and two from Cameroon cluster together with Mansonella perstans with high bootstrap support. Most of the remaining sequences clustered together within the genus Mansonella, but the species could not be resolved. Among the NHP species investigated, a significant association between filarial DNA detection and Oesophagostomum sp. and Necator sp. infection was observed only in gorillas. CONCLUSIONS: To our knowledge, this is the first study reporting DNA from Mansonella spp. in faecal samples. Our results raise questions about the diversity and abundance of these parasites in wildlife, their role as sylvatic reservoirs and their potential for zoonotic transmission. Future studies should focus on detecting variants circulating in both human and NHPs, and improve the molecular information to resolve or support taxonomy classification based on morphological descriptions.

Predisposition to hookworm infection in humans.

Frequency distributions of parasitic helminths within human communities are invariably highly aggregated, the majority of worms occurring in relatively small fractions of the host populations. It has been suggested that the heavily infected individuals are predisposed to this state, not by chance, but by as yet undefined genetic, ecological, behavioral, or social factors. Analyses of individual post-treatment patterns of hookworm reinfection among 112 villagers in an endemic area of West Bengal provide quantitative evidence of predisposition to heavy infection. This observation has implications for the design of control programs based on chemotherapy because of the potential economic advantage of selective or targeted treatment as opposed to mass or blanket treatment.

Terror in the dirt: Sensory determinants of host seeking in soil-transmitted mammalian-parasitic nematodes.

Infection with gastrointestinal parasitic nematodes is a major cause of chronic morbidity and economic burden around the world, particularly in low-resource settings. Some parasitic nematode species, including the human-parasitic threadworm Strongyloides stercoralis and human-parasitic hookworms in the genera Ancylostoma and Necator, feature a soil-dwelling infective larval stage that seeks out hosts for infection using a variety of host-emitted sensory cues. Here, we review our current understanding of the behavioral responses of soil-dwelling infective larvae to host-emitted sensory cues, and the molecular and cellular mechanisms that mediate these responses. We also discuss the development of methods for transgenesis and CRISPR/Cas9-mediated targeted mutagenesis in Strongyloides stercoralis and the closely related rat parasite Strongyloides ratti. These methods have established S. stercoralis and S. ratti as genetic model systems for gastrointestinal parasitic nematodes and are enabling more detailed investigations into the neural mechanisms that underlie the sensory-driven behaviors of this medically and economically important class of parasites.

Molecular features of hookworm larvae (Necator spp.) raised by coproculture from Ugandan chimpanzees and Gabonese gorillas and humans.

Species composition of Necator hookworms was surveyed in (i) Ugandan chimpanzees living around farms and villages at Bulindi, (ii) Gabonese gorillas under habituation in Moukalaba-Doudou National Park (MDNP), and (iii) Gabonese villagers living adjacent to MDNP. Internal transcribed spacers (ITS) of rDNA and partial cytochrome c oxidase subunit 1 (Cox1) gene of mtDNA were analyzed from larvae obtained by coproculture. Three ITS types (I, II and III) and three Cox1 haplotype groups (A, B and C) were demonstrated. ITS type I and Cox1 haplotype group A, representing Necator americanus, were demonstrated in the hookworm larvae from Gabonese gorillas and humans, but not from Ugandan chimpanzees. Type II and haplotype groups B and C, presumably representing N. gorillae, were found in larvae from Ugandan chimpanzees and Gabonese gorillas and humans. These features were overall similar with those found previously in the Central African Republic. Meanwhile, type III was proven in a larva from a Gabonese gorilla as the first demonstration from a non-human primate. Cox1 haplotypes obtained from Ugandan chimpanzees formed a subgroup within group B, presumably reflecting dispersal and diversification processes of the apes.

Dose-ranging study for trials of therapeutic infection with Necator americanus in humans.

Epidemiological studies suggest that a hookworm infection producing 50 eggs/gram of feces may protect against asthma. We conducted a dose-ranging study to identify the dose of hookworm larvae necessary to achieve 50 eggs/gram of feces for therapeutic trials of asthma. Ten healthy subjects without asthma or airway hyperresponsiveness to inhaled methacholine received 10, 25, 50, or 100 Necator americanus larvae administered double blind to an area of skin on the arm. Subjects were seen weekly for 12 weeks and were then treated with mebendazole. Skin itching at the entry site and gastrointestinal symptoms were common at higher doses. Lung function did not change. Levels of blood eosinophils and IgE increased transiently, and levels of IgG increased progressively. All doses resulted in at least 50 eggs/gram of feces in the eight subjects who completed the study. Infection with 10 N. americanus larvae is well tolerated, elicits a modest host eosinophil response, and is potentially suitable for use in preliminary clinical therapeutic trials.

Adult hookworms (Necator spp.) collected from researchers working with wild western lowland gorillas.

BACKGROUND: In general, studies on the diversity of strongylid nematodes in endangered host species are complicated as material obtained by non-invasive sampling methods has limited value for generic and species identification. While egg morphology barely allows assignment to family, the morphology of cultivated infective third stage larvae provides a better resolution at the generic level but cannot be used for exact species identification. Morphology-based taxonomic approaches greatly depend on the examination of adult worms that are usually not available. METHODS: Hookworm parasites in two European researchers, who participated in gorilla research in the Central African Republic, were expelled after anthelmintic treatment to the faeces, collected and morphologically examined. A male worm discharged naturally from a wild bonobo (Pan paniscus) in Congo was also examined for comparison. RESULTS: Two species of Necator were identified in researchers' faecal material: Necator americanus (Stiles, 1902) and N. gorillae Noda & Yamada, 1964; the latter species differed in having a smaller body, smaller buccal cavity and shorter spicules with spade-shaped membranes situated distally. Males of N. gorillae also possessed unusual cuticular thickenings on the dorsal side of the prebursal region of the body. These characters, shared with the male worm from the bonobo, correspond well to the description of N. gorillae described from gorillas in Congo. CONCLUSIONS: Based on the morphology of the hookworms recovered in this study and previous molecular analyses of larvae developed from both humans and western lowland gorillas (Gorilla gorilla gorilla) from this locality, we conclude that the researchers became infected with gorilla hookworms during their stay in the field. This is the first report of infection with a Necator species other than N. americanus in humans.

Schistosoma japonicum and occult blood loss in endemic villages in Leyte, the Philippines.

Schistosoma japonicum has been related to anemia, but the mechanisms mediating this relationship remain unresolved. The primary objective of this study was to assess the role of occult blood loss in mediating S. japonicum-associated anemia after adjusting for age, sex, socioeconomic status (SES), and other helminth infections. The secondary objective was to identify intensity categories of risk for occult blood loss for Trichuris and hookworm after adjustment for the presence of other helminth infections. The role of occult blood loss in mediating S. japonicum-associated anemia was studied cross-sectionally in 729 individuals 8-30 years old in Leyte, The Philippines. Three stool specimens were examined in duplicate for helminth eggs. Hemoglobin, fecal occult blood loss, and anemia were measured and related to the presence and intensity of helminths. Multivariate models were made to adjust for confounding by other helminths and SES. In multivariate models, hemoglobin significantly decreased with increasing infection intensity of S. japonicum, hookworm, and T. trichuria (P < 0.0031, P < 0.0001, and P < 0.0001, respectively). Individuals with higher intensities S. japonicum and T. trichuria were significantly more likely to be fecal occult positive (odds ratio [OR] = 3.54; P = 0.008 and OR = 2.68; P = 0.013, respectively), although this was not true for individuals with hookworm. Additionally, individuals with higher intensities of S. japonicum, hookworm, and T. trichuria were all more likely to be anemic (OR = 3.7, P = 0.0002; OR = 5.3, P = 0.0003; and OR = 1.6, P = 0.021, respectively). It is likely that occult blood loss plays a role only at heavier intensity S. japonicum infections and some other mechanism, such as anemia of inflammation, may be contributing to anemia.

Measurement of anti-enzyme antibodies using an active-site directed radiolabel.

A new technique is described for measuring antibodies to an enzyme which is not available in pure form. The secretions of the rat parasitic nematode, Nippostrongylus brasiliensis, were treated with tritiated diisopropylfluorophosphate. Only one component, an acetylcholinesterase, was radiolabelled. Antibodies to this enzyme in rat antisera were estimated by the Farr technique using the labelled enzyme as antigen. The acetylcholinesterase secreted by Necator americanus, the human hookworm, was similarly specifically labelled.

Cuticle preparations from Necator americanus and their immunogenicity in the infected host.

Cuticle samples have been prepared from the human parasitic nematode Necator americanus using detergent solubilisation with sodium dodecyl sulphate and subsequent reduction of disulphide bonds using 2-beta-mercaptoethanol. It would appear that N. americanus, like many other nematodes, relies on relatively small collagenous proteins, linked by disulphide bonds, to maintain the integrity of its cuticle. These molecules are normally hidden from the immune system during the course of infection but are potentially immunogenic. It is suggested that waves of antibodies, firstly with specificity against superficial cuticular epitopes followed by antibodies against deeper lying, structural elements, should be induced during vaccination to effectively destroy the nematode at the cuticular level.

Identification of hookworm (Necator americanus) antigens and their translation in vitro.

During in vitro culture adult (day 35) Necator americanus synthesise a wide range of protein species many of which are excreted or secreted into the culture medium. Both post infection (day 117) hamster sera and sera from infected humans precipitate antigens of 15, 30, 33, 44, 46 and 69 kDa although individual human sera exhibit some variability in absolute specificity. In immunoblotting experiments antigens of 33 kDa are routinely recognised by human sera although two-dimensional gel analysis suggests that more than one polypeptide is involved. RNA isolated from adult worms direct the in vitro synthesis of numerous polypeptides possessing antigenic determinants recognised by sera from infected hamsters and humans. Post-translational modification of N. americanus encoded polypeptides is not, therefore, a prerequisite for antigenicity.

Filarial parasites exhibit unusually high levels of choline acetyltransferase activity.

The presence of unusually high levels of choline acetyltransferase (ChAT, EC 2.3.1.6) in human and animal filarial parasites has been demonstrated. The levels of ChAT were highest in male worms of Brugia malayi and Brugia pahangi, with specific activities in crude extracts of about 2.27 and 1.26 mumol min-1 (mg protein)-1, respectively. The enzyme levels in these worms were over 10-20 times higher than in male worms of Litomosoides carinii. The ChAT levels were about 2-5 times higher in male than in female worms. The enzyme was also present in appreciably high levels in microfilariae of Brugia species, L. carinii and Wuchereria bancrofti. The levels of ChAT in male worms of Brugia species were several thousand-fold higher than in the intestinal nematodes Trichuris muris and Necator americanus, and were over three orders of magnitude higher than in mammalian brain. Unlike the mammalian ChAT, the parasite enzyme was extremely stable. The parasite enzyme was not inhibited by any of the antifilarial agents except suramin. The filarial ChAT was strongly inhibited by sulphydryl reagents and diethylpyrocarbonate. Ethacrynic acid (EA), a diuretic and a sulphydryl reagent, irreversibly inhibited the filarial ChAT activity at low concentrations. In contrast, EA inhibited the activity of mammalian brain ChAT at much higher concentrations. The motility of adult worms and microfilariae was irreversibly inhibited by low concentrations of EA. Furthermore, the inhibition of motility was paralleled by the inactivation of ChAT in these parasites. These studies indicate that ChAT activity appears to be vital for parasite's survival and that acetylcholine might play a key role in the control of worm motility.

In vitro studies on the relative sensitivity to ivermectin of Necator americanus and Ancylostoma ceylanicum.

Experiments were carried out to compare the sensitivity of Ancylostoma ceylanicum and Necator americanus to ivermectin (IVM) and pyrantel in vitro. Loss of motility and inhibition of ingestion by IVM were compared and A. ceylanicum was found to be approximately 40-50 times more sensitive to IVM than N. americanus. Both species showed a similar sensitivity to pyrantel. Uptake of [3H]IVM across the cuticle was compared and shown to be unlikely to account for the differences in sensitivity to IVM between the two species.