Vorkommen und Übertragung von Simianen Foamy Viren bei wildlebenden Schimpansen

The discovery of the zoonotic potential of retroviruses infecting non-human primates (NHPs) aroused the interest in simian retroviruses such as simian immunodeficiency virus, simian T-cell leukemia virus or simian foamy virus (SFV). However, investigations of retroviral circulation in wild primate communities are rare. Of particular interest are the transmission modalities of SFV, obviously successful strategies, which have led to frequent, persistent infections of NHPs for at least 40 million years. This work is an attempt to determine these modalities in wild chimpanzees from the Taï National Park, Côte d’Ivoire, where SFVs are highly endemic. These analyses presume a fine diagnostic tool to estimate the SFV diversity within each host and identify superinfection cases, i.e., the simultaneous infection of the same individual host with several strains of the same virus. So far, methods possibly allowing such investigations from samples collected non-invasively (such as feces) have never been properly compare. Therefore, the costs and benefits of the gold standard (end-point dilution PCR, EPD-PCR) and multiple bulk-PCR cloning methods were assessed for SFV super-infections based on a case study using fecal samples of two different chimpanzee subspecies ( Pan troglodytes verus and P. t. schweinfurthii ). It could be shown that in these conditions EPD-PCR can lead to massive consumption of biological material. This constitutes a serious drawback in a field in which rarity of biological material is a fundamental constraint. In addition, data demonstrated that EPD- PCR results (single/multiple infection; founder strains) could be well predicted from multiple bulk-PCR clone experiments, by applying simple statistical and network analyses to sequence alignments. Therefore, the implementation of the latter method can be recommended, when the focus is put on retroviral super-infection and only low retroviral loads are encountered. Using this approach, SFV diversity was then estimated for each sample of the study community in Taï National Park to investigate dynamics of SFVs. The results indicate, that vertical transmission (being here understood as mother- offspring transmission) is a common route of SFV transmission within the community whereas previous studies so far only pointed at horizontal transmissions of SFVs. The strong bond between mother and offspring is likely to be responsible for primary infections. With increasing age subsequent infections with SFVs could be observed (super-infections). The development of truly aggressive behavior during the onset of adulthood is hypothesized to result into frequent horizontal transmissions of SFVs between other members of the group. Finally, these data gives evidence for complex SFV dynamics in wild chimpanzees, even at a single community scale, and show that linking wild NHP social interactions and their microorganisms’ dynamics is feasible.Erst die Erkenntnis über das zoonotische Potential von simianen Retroviren, welche nichthumane Primaten (NHP) infizieren, lenkte das wissenschaftliche Interesse auf simiane Retroviren wie das Simiane Immunodefizienz Virus, das Simiane T-cell Leukämie Virus oder das Simiane Foamy Virus (SFV). Dennoch gibt es nur wenige Untersuchungen zur Verbreitung von Retroviren bei wildlebenden Primaten. Von besonderem Interesse sind dabei die Übertragungswege von SFV, welche offenbar eine so erfolgreiche Strategie verfolgen, dass sie bei NHPs seit über 40 Millionen Jahren verbreitet sind und diese persistent infizieren. Die vorliegende Arbeit verfolgt das Ziel die Übertragungswege bei wildlebenden Schimpansen aus dem Taï Nationalpark, Côte d’Ivoire, wo SFV endemisch vorkommt, aufzuzeigen. Solche Analysen setzen jedoch präzise diagnostische Methoden voraus, um die Diversität von SFV innerhalb des Wirtes und dadurch Superinfektionen, i.e., ein Individuum ist parallel mit mehreren Stämmen eines Virus infiziert, zu bestimmen. Die zur Verfügung stehenden Methoden wurden jedoch bisher nicht für den Gebrauch von nicht-invasiv gesammeltem Probenmaterial (z.B. Kotproben) getestet. Daher wurden hier an einem Fallbeispiel mit Kotproben von zwei verschiedenen Schimpansensubspezies Vor- und Nachteile des Goldstandards (Endpunkt-Verdünnungs-PCR, EPDPCR) und der klassischen PCR mit anschließender Klonierung zur Bestimmung von Superinfektionen verglichen. Dabei konnte gezeigt werden, dass unter diesen Konditionen EPD–PCR zu erheblichem Verbrauch von biologischem Material führt. Dies bedeutet eine große Einschränkung in Untersuchungsgebieten, wo biologisches Material nur in begrenztem Maße zur Verfügung steht. Weiterhin zeigen die Daten, dass Ergebnisse der EPD-PCR (Einfach-/ Superinfektion, Identifizierung von Gründersequenzen) auch mittels Experimenten der klassischer PCR und Klonierung erhoben werden können, soweit dabei die Sequenzalignments statistischen und Netzwerkanalysen unterzogen werden. Die Verwendung der letztgenannten Methode kann insbesondere empfohlen werden, wenn retrovirale Superinfektionen im Fokus der Untersuchung stehen und nur eine geringe Viruslast in den Proben vorliegt. Nach diesem Vorgehen wurde im Anschluss die SFV Diversität je Probe erfasst, um die Dynamik von SFV innerhalb der Studiengruppe im Taï National Park zu untersuchen. Im Gegensatz zu bisherigen Studien konnte gezeigt werden, dass die vertikale Übertragung (hier definiert als Mutter-Kind-Übertragung) neben der horizontalen Übertragung eine große Rolle bei der Verbreitung von SFV spielt. Diese Übertragung ist sehr wahrscheinlich für die Primärinfektion mit dem Erreger verantwortlich, da Mutter und Kind zunächst eng miteinander verbunden sind. Mit zunehmendem Alter der Schimpansen wurden jedoch Infektionen mit weiteren SFV Stämmen beobachtet (Superinfektion). Diese sind vermutlich auf horizontale Übertragungen von SFV zwischen Gruppenmitgliedern durch aggressives Verhalten untereinander während des Erwachsenwerdens zurückzuführen. Insgesamt gibt es Hinweise auf eine sehr komplexe Dynamik von Foamy Viren bei wildlebenden Schimpansen bereits auf der Ebene einer einzelnen Schimpansengruppe. Es konnte außerdem gezeigt werden, dass die sozialen Interaktionen des Wirtes Einfluss auf die Dynamik der Mikroorganismen nehmen können

Low dose colonization of broiler chickens with ESBL-/AmpC- producing escherichia coli in a seeder-bird model independent of antimicrobial selection pressure

Extended-spectrum beta-lactamase- (ESBL-) and AmpC beta-lactamase- (AmpC-) producing Enterobacteriaceae pose a risk for both human and animal health. For livestock, highest prevalences have been reported in broiler chickens, which are therefore considered as a reservoir of multidrug-resistant bacteria. The possibility of transfer to humans either by a close contact to colonized broiler flocks or through contaminated retail meat results in the necessity to develop intervention measures for the entire broiler production chain. In this regard, a basic understanding of the colonization process is mandatory including the determination of the minimal bacterial load leading to a persistent colonization of broiler chickens. Therefore, we conducted a bivalent broiler colonization study close to real farming conditions without applying any antimicrobial selection pressure. ESBL- and AmpC- negative broiler chickens (Ross 308) were co- colonized on their third day of life with two strains: one CTX-M-15-producing Escherichia coli-ST410 and one CMY-2/mcr-1-positive E. coli-ST10. Colonization was assessed by cloacal swabs over the period of the trial, starting 24 h post inoculation. During the final necropsy, the contents of crop, jejunum, cecum, and colon were quantified for the occurrence of both bacterial strains. To define the minimal oral colonization dosage 104 to 101 colony forming units (cfu) were orally inoculated to four separately housed broiler groups (each n = 19, all animals inoculated) and a dosage of already 101 cfu E. coli led to a persistent colonization of all animals of the group after 3 days. To assure stable colonization, however, a dosage of 102 cfu E. coli was chosen for the subsequent seeder-bird trial. In the seeder-bird trial one fifth of the animals (seeder, n = 4) were orally inoculated and kept together with the non-inoculated animals (sentinel, n = 16) to mimic the route of natural infection. After 35 days of trial, all animals were colonized with both E. coli strains. Given the low colonization dosage and the low seeder/sentinel ratio, the rapid spread of ESBL- and AmpC- producing Enterobacteriaceae in conventional broiler farms currently seems inevitably resulting in an urgent need for the development of intervention strategies to reduce colonization of broilers during production

Detection of Retroviral Super-Infection from Non-Invasive Samples

While much attention has been focused on the molecular epidemiology of retroviruses in wild primate populations, the correlated question of the frequency and nature of super-infection events, i.e., the simultaneous infection of the same individual host with several strains of the same virus, has remained largely neglected. In particular, methods possibly allowing the investigation of super-infection from samples collected non-invasively (such as faeces) have never been properly compared. Here, we fill in this gap by assessing the costs and benefits of end-point dilution PCR (EPD-PCR) and multiple bulk-PCR cloning, as applied to a case study focusing on simian foamy virus super-infection in wild chimpanzees (Pan troglodytes). We show that, although considered to be the gold standard, EPD-PCR can lead to massive consumption of biological material when only low copy numbers of the target are expected. This constitutes a serious drawback in a field in which rarity of biological material is a fundamental constraint. In addition, we demonstrate that EPD-PCR results (single/multiple infection; founder strains) can be well predicted from multiple bulk-PCR clone experiments, by applying simple statistical and network analyses to sequence alignments. We therefore recommend the implementation of the latter method when the focus is put on retroviral super-infection and only low retroviral loads are encountered

False negativity and positivity as a function of the number of bulk-PCR products analysed.

<p>(a) Probability to detect bimodality (<i>i.e.</i>, super-infection) in the frequency distribution of the number of mismatches per pair of sequences as a function of the number of bulk-PCR products analysed and for subjects for which EPD-PCR revealed a super-infection (n = 6); (b) Probability to detect bimodality in the frequency distribution of the number of mismatches per pair of sequences as a function of the number of bulk-PCR products analysed and for subjects for which EPD-PCR revealed a single infection (n = 4). Shown are median, quartiles, minimum and maximum, of the respective probabilities per subject.</p

Mismatch distribution analyses of EPD-PCR and clone sequence datasets.

<p>Individuals are ordered such that first four individuals with a single infection (as determined by EPD-PCR) are shown (B2, B3, B4 and T3; above the bar) followed by individuals with super-infections (below the bar). Plots are paired for each individual with the EPD-PCR dataset on the left and the bulk-PCR clone dataset on the right. EPD-PCR distributions are black; bulk-PCR clone distributions identified as unimodal (ΔAICc<2) are green; bulk-PCR clone distributions identified as bimodal are purple (ΔAICc>2).</p

Median joining network of bulk PCR product clone sequences.

<p>As in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036570#pone-0036570-g001" target="_blank">Figure 1</a>, the networks are ordered by infection status. Within each network, node size is proportional to the frequency of sequence occurrence (total n = 25 for each individual). Branch lengths are directly related to the number of mutations between sequences, with values noted for differences greater than two base pairs. Clone haplotypes a–f (as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036570#pone-0036570-t001" target="_blank">Table 1</a>) are noted within or adjacent to their corresponding node. Networks generated using TCS were highly similar (data not shown).</p

Alignment of all clone sequences generated in the course of this study

FASTA file comprising 915 SFV int sequences

Retailing Under the N.R.A. II

Data are missing on the diversity of Plasmodium spp. infecting apes that live in their natural habitat, with limited possibility of human-mosquito-ape exchange. We surveyed Plasmodium spp. diversity in wild chimpanzees living in an undisturbed tropical rainforest habitat and found 5 species: P. malariae, P. vivax, P. ovale, P. reichenowi, and P. gaboni