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Campylobacter from contaminated poultry meat is a major source of human gastroenteritis worldwide. To date, attempts to control this zoonotic infection with on-farm biosecurity measures have been inconsistent in outcome. A cornerstone of these efforts has been the detection of chicken infection with microbiological culture, where Campylobacter is generally not detectable until birds are at least 21 days old. Using parallel sequence-based bacterial 16S profiling analysis and targeted sequencing of the porA gene, Campylobacter was identified at very low levels in all commercial flocks at less than 8 days old that were tested from the United Kingdom, Switzerland, and France. These young chicks exhibited a much greater diversity of porA types than older birds testing positive for Campylobacter by culture or quantitative PCR (qPCR). This suggests that as the bacteria multiply sufficiently to be detected by culture methods, one or two variants, as indicated by porA type, dominate the infection. The findings that (i) most young chicks carry some Campylobacter and (ii) not all flocks become Campylobacter positive by culture suggest that efforts to control infection, and therefore avoid contamination of poultry meat, should concentrate on how to limit Campylobacter to low levels by the prevention of the overgrowth of single strains. IMPORTANCE Our results demonstrate the presence of Campylobacter DNA among fecal samples from a range of commercially reared meat chicks that are less than 8 days of age, consistent across 3 European countries. The recently developed, sensitive detection method indicates that infection occurs on commercial farms much earlier and more widely than previously thought, which opens up new opportunities to control Campylobacter contamination at the start of the food chain and reduce the unacceptably high levels of human disease.

Helminth infections are among the World Health Organization’s top neglected diseases with significant impact in many Less Economically Developed Countries. Despite no longer being endemic in Europe, the widespread presence of helminth eggs in archaeological deposits indicates that helminths represented a considerable burden in past European populations. Prevalence of infection is a key epidemiological feature that would influence the elimination of endemic intestinal helminths, for example, low prevalence rates may have made it easier to eliminate these infections in Europe without the use of modern anthelminthic drugs. To determine historical prevalence rates we analysed 589 grave samples from 7 European sites dated between 680 and 1700 CE, identifying two soil transmitted nematodes (Ascaris spp. and Trichuris trichiura) at all locations, and two food derived cestodes (Diphyllobothrium latum and Taenia spp.) at 4 sites. The rates of nematode infection in the medieval populations (1.5 to 25.6% for T. trichiura; 9.3–42.9% for Ascaris spp.) were comparable to those reported within modern endemically infected populations. There was some evidence of higher levels of nematode infection in younger individuals but not at all sites. The genetic diversity of T. trichiura ITS-1 in single graves was variable but much lower than with communal medieval latrine deposits. The prevalence of food derived cestodes was much lower (1.0–9.9%) than the prevalence of nematodes. Interestingly, sites that contained Taenia spp. eggs also contained D. latum which may reflect local culinary practices. These data demonstrate the importance of helminth infections in Medieval Europe and provide a baseline for studies on the epidemiology of infection in historical and modern contexts. Since the prevalence of medieval STH infections mirror those in modern endemic countries the factors affecting STH decline in Europe may also inform modern intervention campaigns., Author summary Parasitic helminths (worms) are important infections of humans in many less well developed countries, particularly those in tropical and sub-tropical regions. These infections are not a major problem in modern Europe but parasite eggs are readily detected in archaeological contexts. To estimate a key epidemiological parameter, the prevalence of infection, we examined large numbers of single graves from Medieval Europe and found that the rates of infection with two soil transmitted nematodes (Ascaris spp. and Trichuris trichiura) were as prevalent as in many modern endemic areas. We also identified two cestodes that humans acquire from eating undercooked red meat (Taenia spp.) or freshwater fish (Diphyllobothrium latum). Using prevalence and ancient DNA data we explored helminth epidemiology in Medieval European populations and factors that may influence infection including age, sex, sanitation, hygiene and culinary practices. The Medieval prevalence rates provide a historical baseline for Europe and an interesting comparator for modern epidemiological studies in other parts of the world. It is noteworthy that helminths were endemic in historical Europe but were eradicated prior to the development of modern drugs. In this sense studying changes in helminth prevalence in historical Europe may provide insights into control efforts in modern endemic regions.

Chicken meat represents an important source of Campylobacter infections of humans world-wide. A better understanding of Campylobacter epidemiology in commercial chicken flocks will facilitate the development of more effective intervention strategies. We developed a gene-specific parallel sequencing approach that efficiently indicated genetic diversity in farm-derived samples and revealed Campylobacter genotypes that would not be detected using microbiological culture. Parallel sequencing of the porA nucleotide fragment identified a different pattern of diversity in broiler flocks compared with broiler-breeder flocks at both individual bird and flock levels. Amongst the flocks tested, broiler flocks and individual birds were dominated by one or two porA fragment types whereas co-dominance with up to six porA fragment types was evident in breeder birds. A high proportion (83.6–93.3%) of porA variants were shared between broiler and breeder flocks. The porA-based diversity profiling could be a useful addition to the repertoire of tools employed to attribute potential sources of contamination for broiler flocks, including the environment, wild animals or other chickens. This approach can be extended to include other loci within Campylobacter and developed for molecular epidemiology studies of other bacterial species.

Throughout history, humans have been afflicted by parasitic worms, and eggs are readily detected in archaeological deposits. This study integrated parasitological and ancient DNA methods with a large sample set dating between Neolithic and Early Modern periods to explore the utility of molecular archaeoparasitology as a new approach to study the past. Molecular analyses provided unequivocal species-level parasite identification and revealed location-specific epidemiological signatures. Faecal–oral transmitted nematodes (Ascaris lumbricoides and Trichuris trichiura) were ubiquitous across time and space. By contrast, high numbers of food-associated cestodes (Diphyllobothrium latum and Taenia saginata) were restricted to medieval Lübeck. The presence of these cestodes and changes in their prevalence at approximately 1300 CE indicate substantial alterations in diet or parasite availability. Trichuris trichiura ITS-1 sequences grouped into two clades; one ubiquitous and one restricted to medieval Lübeck and Bristol. The high sequence diversity of T.t.ITS-1 detected in Lübeck is consistent with its importance as a Hanseatic trading centre. Collectively, these results introduce molecular archaeoparasitology as an artefact-independent source of historical evidence.

The European badger is recognised as a wildlife reservoir for bovine tuberculosis (bTB); the control of which is complex, costly and controversial. Despite the importance of badgers in bTB and the well-documented role for macrophages as anti-mycobacterial effector cells, badger macrophage (bdMφ) responses remain uncharacterised. Here, we demonstrate that bdMφ fail to produce nitric oxide (NO) or upregulate inducible nitric oxide synthase (iNOS) mRNA following Toll-like receptor (TLR) agonist treatment. BdMφ also failed to make NO after stimulation with recombinant badger interferon gamma (bdIFNγ) or a combination of bdIFNγ and lipopolysaccharide. Exposure of bdMφ to TLR agonists and/or bdIFNγ resulted in upregulated cytokine (IL1β, IL6, IL12 and TNFα) mRNA levels indicating that these critical pathways were otherwise intact. Although stimulation with most TLR agonists resulted in strong cytokine mRNA responses, weaker responses were evident after exposure to TLR9 agonists, potentially due to very low expression of TLR9 in bdMφ. Both NO and TLR9 are important elements of innate immunity to mycobacteria, and these features of bdMφ biology would impair their capacity to resist bTB infection. These findings have significant implications for the development of bTB management strategies, and support the use of vaccination to reduce bTB infection in badgers.

Hard ticks subvert the immune responses of their vertebrate hosts in order to feed for much longer periods than other blood-feeding ectoparasites; this may be one reason why they transmit perhaps the greatest diversity of pathogens of any arthropod vector. Tick-induced immunomodulation is mediated by salivary components, some of which neutralise elements of innate immunity or inhibit the development of adaptive immunity. As dendritic cells (DC) trigger and help to regulate adaptive immunity, they are an ideal target for immunomodulation. However, previously described immunoactive components of tick saliva are either highly promiscuous in their cellular and molecular targets or have limited effects on DC. Here we address the question of whether the largest and globally most important group of ticks (the ixodid metastriates) produce salivary molecules that specifically modulate DC activity. We used chromatography to isolate a salivary gland protein (Japanin) from Rhipicephalus appendiculatus ticks. Japanin was cloned, and recombinant protein was produced in a baculoviral expression system. We found that Japanin specifically reprogrammes DC responses to a wide variety of stimuli in vitro, radically altering their expression of co-stimulatory and co-inhibitory transmembrane molecules (measured by flow cytometry) and their secretion of pro-inflammatory, anti-inflammatory and T cell polarising cytokines (assessed by Luminex multiplex assays); it also inhibits the differentiation of DC from monocytes. Sequence alignments and enzymatic deglycosylation revealed Japanin to be a 17.7 kDa, N-glycosylated lipocalin. Using molecular cloning and database searches, we have identified a group of homologous proteins in R. appendiculatus and related species, three of which we have expressed and shown to possess DC-modulatory activity. All data were obtained using DC generated from at least four human blood donors, with rigorous statistical analysis. Our results suggest a previously unknown mechanism for parasite-induced subversion of adaptive immunity, one which may also facilitate pathogen transmission., Author Summary Dendritic cells (DC) are specialised cells of the vertebrate immune system. DC can sense different types of infectious agents and parasites, and both trigger and help regulate the specific types of immunity needed to eliminate them. We have discovered that the largest and globally most important group of hard ticks produce a unique family of proteins in their saliva that selectively targets DC, radically altering functions that would otherwise induce robust immune responses; these proteins also prevent DC developing from precursor cells. The production of these salivary molecules may help to explain two highly unusual features of these hard ticks compared with other blood-feeding parasites: their ability to feed continuously on their vertebrate hosts for considerable lengths of time (7 days or more) without eliciting potentially damaging immune responses, and their capacity to transmit possibly the greatest variety of pathogens of any type of invertebrate.

The incorporation of RANTES or IL-23 into DNA vaccines may improve their immunogenicity by the recruitment and activation of dendritic cells. This may also select for a TH1 response counteracting the TH2 response which can predominate when a DNA vaccine is delivered by gene gun. We have immunized mice with various DNA constructs encoding APR/8/34 influenza virus hemagglutinin (HA), either fused to or separate from, IL-23 or RANTES using a gene gun. Those immunized with IL-23/HA fusion constructs and challenged with influenza 27 weeks post-vaccination, tended to have cleared more virus than those vaccinated with HA DNA. Mice immunized with the RANTES/HA fusion construct produced a mixed TH1/TH2 response whereas in HA-vaccinated mice, a TH2 response predominated. Immunization with a plasmid in which HA and RANTES were under the control of separate promoters, failed to generate a mixed TH1/TH2 response suggesting that enhanced antigen uptake via RANTES receptors may contribute to the mixed immune response generated to the fusion construct. Overall these findings provide further evidence that Type 1 cytokines or chemokines, fused to antigen in a DNA vaccine, can influence the nature and the longevity of the immune response and ultimately, its protective capacity.