Experimental evolution of infectious behaviour in a facultative ectoparasite

Parasitic lifestyles have evolved many times in animals, but how such life‐history strategies evolved from free‐living ancestors remains a great puzzle. Transitional symbiotic strategies, such as facultative parasitism, are hypothesized evolutionary stepping stones towards obligate parasitism. However, to consider this hypothesis, heritable genetic variation in infectious behaviour of transitional symbiotic strategies must exist. In this study, we experimentally evolved infectivity and estimated the additive genetic variation in a facultative parasite. We performed artificial selection experiments in which we selected for either increased or decreased propensity to infect in a facultatively parasitic mite (Macrocheles muscaedomesticae). Here, infectiousness was expressed in terms of mite attachment to a host (Drosophila hydei) and modelled as a threshold trait. Mites responded positively to selection for increased infectivity; realized heritability of infectious behaviour was significantly different from zero and estimated to be 16.6% (±4.4% SE). Further, infection prevalence was monitored for 20 generations post‐selection. Selected lines continued to display relatively high levels of infection, demonstrating a degree of genetic stability in infectiousness. Our study is the first to provide an estimate of heritability and additive genetic variation for infectious behaviour in a facultative parasite, which suggests natural selection can act upon facultative strategies with important implications for the evolution of parasitism.     

Circannual variation in blood parasitism in a sub‐Saharan migrant passerine bird,the garden warbler

Knowing the natural dynamics of pathogens in migratory birds is important, for example, to understand the factors that influence the transport of pathogens to and their transmission in new geographical areas, whereas the transmission of other pathogens might be restricted to a specific area. We studied haemosporidian blood parasites of the genera Plasmodium, Haemoproteus and Leucocytozoon in a migratory bird, the garden warbler Sylvia borin. Birds were sampled in spring, summer and early autumn at breeding grounds in Sweden, on migration at Capri, Italy and on arrival and departure from wintering staging areas in West Africa: mapping recoveries of garden warblers ringed in Fennoscandia and Capri showed that these sites are most probably on the migratory flyway of garden warblers breeding at Kvismaren. Overall, haemosporidian prevalence was 39%, involving 24 different parasite lineages. Prevalence varied significantly over the migratory cycle, with relatively high prevalence of blood parasites in the population on breeding grounds and at the onset of autumn migration, followed by marked declines in prevalence during migration both on spring and autumn passage. Importantly, we found that when examining circannual variation in the different lineages, significantly different prevalence profiles emerged both between and within genera. Our results suggest that differences in prevalence profiles are the result of either different parasite transmission strategies or coevolution between the host and the various parasite lineages. When separating parasites into common vs. rare lineages, we found that two peaks in the prevalence of rare parasites occur; on arrival at Swedish breeding grounds, and after the wintering period in Africa. Our results stress the importance of appropriate taxonomic resolution when examining host‐parasite interactions, as variation in prevalence both between and within parasite genera can show markedly different patterns.     

A genome‐wide survey reveals abundant rice blast R genes in resistant cultivars

Plant resistance genes (R genes) harbor tremendous allelic diversity, constituting a robust immune system effective against microbial pathogens. Nevertheless, few functional R genes have been identified for even the best‐studied pathosystems. Does this limited repertoire reflect specificity, with most R genes having been defeated by former pests, or do plants harbor a rich diversity of functional R genes, the composite behavior of which is yet to be characterized? Here, we survey 332 NBS‐LRR genes cloned from five resistant Oryza sativa (rice) cultivars for their ability to confer recognition of 12 rice blast isolates when transformed into susceptible cultivars. Our survey reveals that 48.5% of the 132 NBS‐LRR loci tested contain functional rice blast R genes, with most R genes deriving from multi‐copy clades containing especially diversified loci. Each R gene recognized, on average, 2.42 of the 12 isolates screened. The abundant R genes identified in resistant genomes provide extraordinary redundancy in the ability of host genotypes to recognize particular isolates. If the same is true for other pathogens, many extant NBS‐LRR genes retain functionality. Our success at identifying rice blast R genes also validates a highly efficient cloning and screening strategy.     

Current advances in aptamer‐assisted technologies for detecting bacterial and fungal toxins

Infectious diseases are among the common leading causes of morbidity and mortality worldwide. Associated with the emergence of new infectious diseases, the increasing number of antimicrobial‐resistant isolates presents a serious threat to public health and hospitalized patients. A microbial pathogen may elicit several host responses and use a variety of mechanisms to evade host defences. These methods and mechanisms include capsule, lipopolysaccharides or cell wall components, adhesions and toxins. Toxins inhibit phagocytosis, cause septic shock and host cell damages by binding to host surface receptors and invasion. Bacterial and fungal pathogens are able to apply many different toxin‐dependent mechanisms to disturb signalling pathways and the structural integrity of host cells for establishing and maintaining infections Initial techniques for analysis of bacterial toxins were based on in vivo or in vitro assessments. There is a permanent demand for appropriate detection methods which are affordable, practical, careful, rapid, sensitive, efficient and economical. Aptamers are DNA or RNA oligonucleotides that are selected by systematic evolution of ligands using exponential enrichment (SELEX) methods and can be applied in diagnostic applications. This review provides an overview of aptamer‐based methods as a novel approach for detecting toxins in bacterial and fungal pathogens.     

Ecological and evolutionary drivers of haemoplasma infection and bacterial genotype sharing in a Neotropical bat community

Most emerging pathogens can infect multiple species, underlining the importance of understanding the ecological and evolutionary factors that allow some hosts to harbour greater infection prevalence and share pathogens with other species. However, our understanding of pathogen jumps is based primarily around viruses, despite bacteria accounting for the greatest proportion of zoonoses. Because bacterial pathogens in bats (order Chiroptera) can have conservation and human health consequences, studies that examine the ecological and evolutionary drivers of bacterial prevalence and barriers to pathogen sharing are crucially needed. Here were studied haemotropic Mycoplasma spp. (i.e., haemoplasmas) across a species‐rich bat community in Belize over two years. Across 469 bats spanning 33 species, half of individuals and two‐thirds of species were haemoplasma positive. Infection prevalence was higher for males and for species with larger body mass and colony sizes. Haemoplasmas displayed high genetic diversity (21 novel genotypes) and strong host specificity. Evolutionary patterns supported codivergence of bats and bacterial genotypes alongside phylogenetically constrained host shifts. Bat species centrality to the network of shared haemoplasma genotypes was phylogenetically clustered and unrelated to prevalence, further suggesting rare—but detectable—bacterial sharing between species. Our study highlights the importance of using fine phylogenetic scales when assessing host specificity and suggests phylogenetic similarity may play a key role in host shifts not only for viruses but also for bacteria. Such work more broadly contributes to increasing efforts to understand cross‐species transmission and the epidemiological consequences of bacterial pathogens.     

Effective population size as a driver for divergence of an antimicrobial peptide (Hymenoptaecin) in two common European bumblebee species

Social insects are the target of numerous pathogens. This is because the high density of closely‐related individuals frequently interacting with each other enhances the transmission and establishment of pathogens. This high selective pressure results in the rapid evolution of immune genes, which might be counteracted by a reduced effective population size (N_e) lowering the effectiveness of selection. We tested the effect of N_e on the evolutionary rate of an important immune gene for the antimicrobial peptide Hymenoptaecin in two common central European bumblebee species: Bombus terrestris and Bombus lapidarius. Both species are similar in their biology and are expected to be under similar selective pressures because pathogen prevalence does not differ between species. However, previous studies indicated a higher N_e in B. terrestris compared to B. lapidarius. We found high intraspecific variability in the coding sequence but low variability for silent polymorphisms in B. lapidarius. Estimates of long‐ and short‐term N_e were three‐ to four‐fold higher N_e in B. terrestris, although the species did not differ in census population sizes. The difference in N_e might result in less efficient selection and suboptimal adaptation of immune genes (e.g. hymenoptaecin) in B. lapidarius, and thus this species might become less resistant and more tolerant, turning into a superspreader of diseases.     

Knock‐on community impacts of a novel vector: spillover of emerging DWV‐B from Varroa‐infested honeybees to wild bumblebees

Novel transmission routes can directly impact the evolutionary ecology of infectious diseases, with potentially dramatic effect on host populations and knock‐on effects on the wider host community. The invasion of Varroa destructor, an ectoparasitic viral vector in Western honeybees, provides a unique opportunity to examine how a novel vector affects disease epidemiology in a host community. This specialist honeybee mite vectors deformed wing virus (DWV), an important re‐emerging honeybee pathogen that also infects wild bumblebees. Comparing island honeybee and wild bumblebee populations with and without V. destructor, we show that V. destructor drives DWV prevalence and titre in honeybees and sympatric bumblebees. Viral genotypes are shared across hosts, with the potentially more virulent DWV‐B overtaking DWV‐A in prevalence in a current epidemic. This demonstrates disease emergence across a host community driven by the acquisition of a specialist novel transmission route in one host, with dramatic community level knock‐on effects.     

Avian Plasmodium lineages found in spot surveys of mosquitoes from 2007 to 2010 at Sakata wetland,Japan: do dominant lineages persist for multiple years?

The ecology and geographical distribution of disease vectors are major determinants of spatial and temporal variations in the transmission dynamics of vector‐borne pathogens. However, there are limited studies on the ecology of vectors that contribute to the natural transmission of most vector‐borne pathogens. Avian Plasmodium parasites are multihost mosquito‐borne pathogens transmitted by multiple mosquito species, which might regulate the diversity and persistence of these parasites. From 2007 to 2010, we conducted entomological surveys at Sakata wetland in central Japan, to investigate temporal variation in mosquito occurrence and prevalence of avian Plasmodium lineages in the mosquito populations. A polymerase chain reaction (PCR)‐based method was used to detect Plasmodium parasites and identify the blood sources of mosquitoes. Culex inatomii and C. pipiens pallens represented 60.0% and 34.8% of 11 mosquito species collected, respectively. Our results showed that the two dominant mosquito species most likely serve as principal vectors of avian Plasmodium parasites during June, which coincides with the breeding season of bird species nesting in the wetland reed beds. Fourteen animal species were identified as blood sources of mosquitoes, with the oriental reed warbler (Acrocephalus orientalis) being the commonest blood source. Although there was significant temporal variation in the occurrence of mosquitoes and prevalence of Plasmodium lineages in the mosquitoes, the dominant Plasmodium lineages shared by the two dominant mosquito species were consistently found at the same time during transmission seasons. Because vector competence cannot be confirmed solely by PCR approaches, experimental demonstration is required to provide definitive evidence of transmission suggested in this study.     

Grapevine VpPR10.1 functions in resistance to Plasmopara viticola through triggering a cell death‐like defence response by interacting with VpVDAC3

As one of the most serious diseases in grape, downy mildew caused by Plasmopara viticola is a worldwide grape disease. Much effort has been focused on improving susceptible grapevine resistance, and wild resistant grapevine species are important for germplasm improvement of commercial cultivars. Using yeast two‐hybrid screen followed by a series of immunoprecipitation experiments, we identified voltage‐dependent anion channel 3 (VDAC3) protein from Vitis piasezkii ‘Liuba‐8’ as an interacting partner of VpPR10.1 cloned from Vitis pseudoreticulata ‘Baihe‐35‐1’, which is an important germplasm for its resistance to a range of pathogens. Co‐expression of VpPR10.1/VpVDAC3 induced cell death in Nicotiana benthamiana, which accompanied by ROS accumulation. VpPR10.1 transgenic grapevine line showed resistance to P. viticola. We conclude that the VpPR10.1/VpVDAC3 complex is responsible for cell death‐mediated defence response to P. viticola in grapevine.     

Antimicrobial resistance and toxin gene profiles of Staphylococcus aureus strains from Holstein milk

Isolation of Staphylococcus aureus (Staph. aureus) from Holstein milk samples with mastitis and nonmastitis was conducted to estimate its prevalence, antimicrobial resistance and toxin genes. A total of 353 milk samples were collected from three Chinese Holstein herds. Fifty‐three Staph. aureus isolates collected from 29 Staph. aureus‐positive samples were characterized via antimicrobial susceptibility, toxin genes and Pulsed‐field Gel Electrophoresis (PFGE) profiles. The prevalence of Staph. aureus was 4·0–9·5% in mastitic and 7·3–11·5% in nonmastitic samples in the analysed herds. Approximately 61·0% of Staph. aureus strains isolated from mastitis cows were resistant to ≥10 antimicrobials compared with 0% of isolates with nonmastitis. The most frequently observed super antigenic toxin gene was pvl (41·5%) followed by seh + pvl (13·2%). We did not find mecA‐positive methicillin‐resistant Staph. aureus (MRSA) strains, while mecA‐negative MRSA strains were identified in the three herds. PFGE results suggested potential transmission of Staph. aureus strains in different farms. These results open new insights into Staph. aureus transmission and antimicrobial resistance of Holstein dairy cows and into developing strategies for udder health improvement of dairy cattle.     

Are we overestimating risk of enteric pathogen spillover from wild birds to humans?

Enteric illnesses remain the second largest source of communicable diseases worldwide, and wild birds are suspected sources for human infection. This has led to efforts to reduce pathogen spillover through deterrence of wildlife and removal of wildlife habitat, particularly within farming systems, which can compromise conservation efforts and the ecosystem services wild birds provide. Further, Salmonella spp. are a significant cause of avian mortality, leading to additional conservation concerns. Despite numerous studies of enteric bacteria in wild birds and policies to discourage birds from food systems, we lack a comprehensive understanding of wild bird involvement in transmission of enteric bacteria to humans. Here, we propose a framework for understanding spillover of enteric pathogens from wild birds to humans, which includes pathogen acquisition, reservoir competence and bacterial shedding, contact with people and food, and pathogen survival in the environment. We place the literature into this framework to identify important knowledge gaps. Second, we conduct a meta‐analysis of prevalence data for three human enteric pathogens, Campylobacter spp., E. coli, and Salmonella spp., in 431 North American breeding bird species. Our literature review revealed that only 3% of studies addressed the complete system of pathogen transmission. In our meta‐analysis, we found a Campylobacter spp. prevalence of 27% across wild birds, while prevalence estimates of pathogenic E. coli (20%) and Salmonella spp. (6.4%) were lower. There was significant bias in which bird species have been tested, with most studies focusing on a small number of taxa that are common near people (e.g. European starlings Sturnus vulgaris and rock pigeons Columba livia) or commonly in contact with human waste (e.g. gulls). No pathogen prevalence data were available for 65% of North American breeding bird species, including many commonly in contact with humans (e.g. black‐billed magpie Pica hudsonia and great blue heron Ardea herodias), and our metadata suggest that some under‐studied species, taxonomic groups, and guilds may represent equivalent or greater risk to human infection than heavily studied species. We conclude that current data do not provide sufficient information to determine the likelihood of enteric pathogen spillover from wild birds to humans and thus preclude management solutions. The primary focus in the literature on pathogen prevalence likely overestimates the probability of enteric pathogen spillover from wild birds to humans because a pathogen must survive long enough at an infectious dose and be a strain that is able to colonize humans to cause infection. We propose that future research should focus on the large number of under‐studied species commonly in contact with people and food production and demonstrate shedding of bacterial strains pathogenic to humans into the environment where people may contact them. Finally, studies assessing the duration and intensity of bacterial shedding and survival of bacteria in the environment in bird faeces will help provide crucial missing information necessary to calculate spillover probability. Addressing these essential knowledge gaps will support policy to reduce enteric pathogen spillover to humans and enhance bird conservation efforts that are currently undermined by unsupported fears of pathogen spillover from wild birds.     

Reappraising plastid markers of the red algae for phylogenetic community ecology in the genomic era

Selection of appropriate genetic markers to quantify phylogenetic diversity is crucial for community ecology studies. Yet, systematic evaluation of marker genes for this purpose is scarcely done. Recently, the combined effort of phycologists has produced a rich plastid genome resource with taxonomic representation spanning all of the major lineages of the red algae (Rhodophyta). In this proof‐of‐concept study, we leveraged this resource by developing and applying a phylogenomic strategy to seek candidate plastid markers suitable for phylogenetic community analysis. We ranked the core genes of 107 published plastid genomes based on various sequence‐derived properties and their tree distance to plastid genome phylogenies. The resulting ranking revealed that the most widely used marker, rbcL, is not necessarily the optimal marker, while other promising markers might have been overlooked. We designed and tested PCR primers for several candidate marker genes, and successfully amplified one of them, rpoC1, in a taxonomically broad set of red algal specimens. We suggest that our general marker identification methodology and the rpoC1 primers will be useful to the phycological community for investigating the biodiversity and community ecology of the red algae.     

Helicobacter pylori recrudescence and its influencing factors

Helicobacter pylori (H pylori) is known as one of the most common infectious pathogens, with high infection and recurrence rates worldwide. The prevalence of H pylori is up to 90% in developing countries, while the annual recurrence rate is much higher than that in developed countries. Recurrence can occur either by recrudescence or reinfection. Compared with reinfection, the time window for recrudescence is generally shorter, followed by the recurrence of H pylori–associated diseases in the short‐term. Many factors are involved in the H pylori reinfection, such as the prevalence of H pylori infection, living conditions and economic development, health conditions and so forth. Previous studies focused less on H pylori recrudescence. Therefore, the influencing factors for H pylori recrudescence needed further exploration. This study reviewed the recrudescence of H pylori infection and its influencing factors.     

Vying for the control of inflammasomes: The cytosolic frontier of enteric bacterial pathogen–host interactions

Enteric pathogen–host interactions occur at multiple interfaces, including the intestinal epithelium and deeper organs of the immune system. Microbial ligands and activities are detected by host sensors that elicit a range of immune responses. Membrane‐bound toll‐like receptors and cytosolic inflammasome pathways are key signal transducers that trigger the production of pro‐inflammatory molecules, such as cytokines and chemokines, and regulate cell death in response to infection. In recent years, the inflammasomes have emerged as a key frontier in the tussle between bacterial pathogens and the host. Inflammasomes are complexes that activate caspase‐1 and are regulated by related caspases, such as caspase‐11, ‐4, ‐5 and ‐8. Importantly, enteric bacterial pathogens can actively engage or evade inflammasome signalling systems. Extracellular, vacuolar and cytosolic bacteria have developed divergent strategies to subvert inflammasomes. While some pathogens take advantage of inflammasome activation (e.g. Listeria monocytogenes, Helicobacter pylori), others (e.g. E. coli, Salmonella, Shigella, Yersinia sp.) deploy a range of virulence factors, mainly type 3 secretion system effectors, that subvert or inhibit inflammasomes. In this review we focus on inflammasome pathways and their immune functions, and discuss how enteric bacterial pathogens interact with them. These studies have not only shed light on inflammasome‐mediated immunity, but also the exciting area of mammalian cytosolic immune surveillance.     

Fast Technology Analysis Enables Identification of Species and Genotypes of Latent Microsporidia Infections in Healthy Native Cameroonians

Several enteric microsporidia species have been detected in humans and other vertebrates and their identifications at the genotype level are currently being elucidated. As advanced methods, reagents, and disposal kits for detecting and identifying pathogens become commercially available, it is important to test them in settings other than in laboratories with “state‐of‐the‐art” equipment and well‐trained staff members. In the present study, we sought to detect microsporidia DNA preserved and extracted from FTA (fast technology analysis) cards spotted with human fecal suspensions obtained from Cameroonian volunteers living in the capital city of Yaoundé to preclude the need for employing spore‐concentrating protocols. Further, we tested whether amplicon nucleotide sequencing approaches could be used on small aliquots taken from the cards to elucidate the diversity of microsporidia species and strains infecting native residents. Of 196 samples analyzed, 12 (6.1%) were positive for microsporidia DNA; Enterocytozoon bieneusi (Type IV and KIN‐1), Encephalitozoon cuniculi, and Encephalitozoon intestinalis were identified. These data demonstrate the utility of the FTA cards in identifying genotypes of microsporidia DNA in human fecal samples that may be applied to field testing for prevalence studies.     

Genotypic variation in Norway spruce correlates to fungal communities in vegetative buds

The taxonomically diverse phyllosphere fungi inhabit leaves of plants. Thus, apart from the fungi's dispersal capacities and environmental factors, the assembly of the phyllosphere community associated with a given host plant depends on factors encoded by the host's genome. The host genetic factors and their influence on the assembly of phyllosphere communities under natural conditions are poorly understood, especially in trees. Recent work indicates that Norway spruce (Picea abies) vegetative buds harbour active fungal communities, but these are hitherto largely uncharacterized. This study combines internal transcribed spacer sequencing of the fungal communities associated with dormant vegetative buds with a genome‐wide association study (GWAS) in 478 unrelated Norway spruce trees. The aim was to detect host loci associated with variation in the fungal communities across the population, and to identify loci correlating with the presence of specific, latent, pathogens. The fungal communities were dominated by known Norway spruce phyllosphere endophytes and pathogens. We identified six quantitative trait loci (QTLs) associated with the relative abundance of the dominating taxa (i.e., top 1% most abundant taxa). Three additional QTLs associated with colonization by the spruce needle cast pathogen Lirula macrospora or the cherry spruce rust (Thekopsora areolata) in asymptomatic tissues were detected. The identification of the nine QTLs shows that the genetic variation in Norway spruce influences the fungal community in dormant buds and that mechanisms underlying the assembly of the communities and the colonization of latent pathogens in trees may be uncovered by combining molecular identification of fungi with GWAS.     

Development of a bioassay to assess resistance to Fusarium oxysporum (Schlecht.) in asparagus (Asparagus officinalis L.)

Fusarium oxysporum is one of the major pathogens causing root and crown rot in asparagus. Breeding of cultivars resistant to F. oxysporum would be the most efficient strategy for pathogen control. In this study, a bioassay was developed for screening seedling resistance. The non‐destructive bioassay comprises inoculation with a highly aggressive F. oxysporum isolate, incubation in a climate chamber and quantification of disease symptoms by a digital image analysing system and a PTA‐ELISA. This bioassay is simple to implement and demonstrated high reproducibility. Subsequently, it was used to determine the resistance behaviour of 16 asparagus genotypes to F. oxysporum. The asparagus cultivars revealed different levels of susceptibility, whereas the wild relative A. densiflorus was confirmed to be resistant.     

The 12th International Workshops on Opportunistic Protists (IWOP‐12)

The 12th International Workshops on Opportunistic Protists (IWOP‐12) was held in August 2012 in Tarrytown, New York. The objectives of the IWOP meetings are to: (1) serve as a forum for exchange of new information among active researchers concerning the basic biology, molecular genetics, immunology, biochemistry, pathogenesis, drug development, therapy, and epidemiology of these immunodeficiency‐associated pathogenic eukaryotic microorganisms that are seen in patients with AIDS and (2) foster the entry of new and young investigators into these underserved research areas. The IWOP meeting focuses on opportunistic protists, e.g. the free‐living amoebae, Pneumocystis, Cryptosporidium, Toxoplasma, the Microsporidia, and kinetoplastid flagellates. This conference represents the major conference that brings together research groups working on these opportunistic pathogens. Slow but steady progress is being achieved on understanding the biology of these pathogenic organisms, their involvement in disease causation in both immune‐deficient and immune‐competent hosts, and is providing critical insights into these emerging and reemerging pathogens. This IWOP meeting demonstrated the importance of newly developed genomic level information for many of these pathogens and how analysis of such large data sets is providing key insights into the basic biology of these organisms. A great concern is the loss of scientific expertise and diversity in the research community due to the ongoing decline in research funding. This loss of researchers is due to the small size of many of these research communities and a lack of appreciation by the larger scientific community concerning the state of art and challenges faced by researchers working on these organisms.