An integrated approach to pathogen transmission via environmental reservoirs

To mitigate the effects of zoonotic diseases on human and animal populations, it is critical to understand what factors alter transmission dynamics. Here we assess the risk of exposure to lethal concentrations of the anthrax bacterium, Bacillus anthracis, for grazing animals in a natural system over time through different transmission mechanisms. We follow pathogen concentrations at anthrax carcass sites and waterholes for five years and estimate infection risk as a function of grass, soil or water intake, age of carcass sites, and the exposure required for a lethal infection. Grazing, not drinking, seems the dominant transmission route, and transmission is more probable from grazing at carcass sites 1–2 years of age. Unlike most studies of virulent pathogens that are conducted under controlled conditions for extrapolation to real situations, we evaluate exposure risk under field conditions to estimate the probability of a lethal dose, showing that not all reservoirs with detectable pathogens are significant transmission pathways

Distribution and Molecular Evolution of Bacillus anthracis Genotypes in Namibia

The recent development of genetic markers for Bacillus anthracis has made it possible to monitor the spread and distribution of this pathogen during and between anthrax outbreaks. In Namibia, anthrax outbreaks occur annually in the Etosha National Park (ENP) and on private game and livestock farms. We genotyped 384 B. anthracis isolates collected between 1983–2010 to identify the possible epidemiological correlations of anthrax outbreaks within and outside the ENP and to analyze genetic relationships between isolates from domestic and wild animals. The isolates came from 20 animal species and from the environment and were genotyped using a 31-marker multi-locus-VNTR-analysis (MLVA) and, in part, by twelve single nucleotide polymorphism (SNP) markers and four single nucleotide repeat (SNR) markers. A total of 37 genotypes (GT) were identified by MLVA, belonging to four SNP-groups. All GTs belonged to the A-branch in the cluster- and SNP-analyses. Thirteen GTs were found only outside the ENP, 18 only within the ENP and 6 both inside and outside. Genetic distances between isolates increased with increasing time between isolations. However, genetic distance between isolates at the beginning and end of the study period was relatively small, indicating that while the majority of GTs were only found sporadically, three genetically close GTs, accounting for more than four fifths of all the ENP isolates, appeared dominant throughout the study period. Genetic distances among isolates were significantly greater for isolates from different host species, but this effect was small, suggesting that while species-specific ecological factors may affect exposure processes, transmission cycles in different host species are still highly interrelated. The MLVA data were further used to establish a model of the probable evolution of GTs within the endemic region of the ENP. SNR-analysis was helpful in correlating an isolate with its source but did not elucidate epidemiological relationships

GIS-based distribution analysis of Bacillus anthracis in Etosha National Park and farms with wildlife and livestock in Namibia using molecular- epidemiological methods

Milzbrand (Anthrax), als `Neglected Zoonotic Disease`, führt in Entwicklungsländern immer noch zu hoher Mortalität bei Mensch und Tier. Das Auftreten dieser Erkrankung bei Wild- und Nutztieren ist vor allem im südlichen Afrika endemisch und sorgt im Etosha Nationalpark (ENP) in Namibia regelmäßig für vermehrte Fälle. In dieser Arbeit wurde das Auftreten von Anthrax im ENP vor dem Hintergrund epidemiologischer Zusammenhänge untersucht und mit vorliegenden Studien aus anderen Nationalparks verglichen. Mittels molekularbiologischer Verfahren wurde die Diversität des Erregers be-stimmt, um durch Clusteranalysen der Genotypen (GT) Aufschluss über ihre räumliche und zeitliche Verteilung und das Ausbreitungsmuster im ENP zu erhalten. Es sollte geprüft wer-den, in wie weit diese Verfahren als unterstützendes Mittel zur Aufklärung epidemiologischer Zusammenhänge von gehäuften Anthraxfällen sinnvoll eingesetzt werden können. Unter-sucht wurden Proben aus Kadavern gefallener Tiere und Proben von Umwelthabitaten des ENP. B. anthracis-Isolate wurden nach dem von Keim et al. (2004) vorgeschlagenem PHRANA-System genotypisiert. Nach SNP-Analyse von 525 B. anthracis-Isolaten aus Gesamt- Namibia ergaben sich vier phylogenetische Gruppen, die auf vier unterschiedliche Einträge nach Namibia schließen lassen (Beyer et al., 2012). Die MLVA 31 Marker ergab 44 GT, 24 GT davon wurden ausschließlich im ENP nachgewiesen. Die von fünf GT bisher durchgeführten SNR-Analysen von 268 Isolaten ergaben 53 SNR-Typen. Die Ergebnisse der MLVA 31 Marker zeigten, dass das Auftreten von Anthrax durch unterschiedliche GT verursacht wurde, die bei einzelnen Spezies (Zebra und Springbock) signifikant häufiger als bei anderen Wildtierspezies nachgewiesen wurden. Ebenso wurden Regionen im Park identifiziert, in denen signifikant gehäuft bestimmte GT isoliert wurden. Räumliche und spezies-spezifische Nachweise von GT wurde auf Standorte der jeweiligen Wildspezies zurückgeführt und durch Ergebnisse der Korrelation zwischen GT und Bodentypen bzw. Vegetationszonen bestätigt. Der Vergleich der Cluster positiver Nachweise mit GT-Clustern im Park zeigte, entgegen der Erwartung, ein deutlich kleineres Cluster für den am häufigsten nachgewiesenen GT (53% der ENP-Isolate) im Vergleich zum zweit häufigsten GT (22% der ENP- Isolate). Die GT-Cluster deckten sich mit dem Cluster positiver Anthrax- Nachweise und lieferten keine weiteren Aussagen zum möglichen Anthrax- Ausbreitungsmuster. Signifikant vermehrt positive Nachweise bei Tierproben fanden sich für die Periode 1983-2011 zwar in der Regenzeit. Bei gleichmäßiger Surveillance während der Regen- und Trockenzeit (2009/2010) zeigte sich allerdings kein signifikanter Zusammenhang zwischen dem Ereignis Regen und vermehrt positiven Nachweisen. Lindeques (1991) Ergebnisse zum saisonalen Auftreten vermehrt positiver Nachweise bei einzelnen Spezies konnten nur teilweise bestätigt werden. Die Langzeitstudie der Umweltproben bestätigte für die untersuchten Einteilungskategorien, mit einer Ausnahme, die „Inkubator“-Theorie für „Risiko-Habitate“. Zur Untermauerung der sich abzeichnenden Tendenz wären jedoch weitere, einen noch längeren Zeitraum abdeckende Untersuchungen erforderlich. Da zwar signifikant vermehrt positive Ergebnisse von B. anthracis in Umweltproben in der Trockenzeit mit im Vergleich zur Regenzeit jedoch geringeren Konzentrationen vorlagen, könnte ein jahreszeitenabhängiges Infektionsrisiko mit erhöhtem Risiko während der Regenzeit abgeleitet werden. Doppelinfektionen traten nach Feststellung von sich räumlich überlappenden Clustern involvierter GT nicht unerwartet auf. Nach Detektion mehrerer GT aus teilweise derselben Wasserstelle und dem Vergleich der GT aus Tier- und Umweltproben können Wasserstellen als potentielle Infektionsquellen nicht ausgeschlossen werden.Anthrax, one of the Neglected Zoonotic Diseases, still remains a major cause of high mortalities in humans and animals in developing countries. The disease is endemic in southern Africa in domestic animals and wildlife and occurs regularly in the Etosha National Park (ENP) in Namibia. This dissertation presents results of a molecular-epidemiological study using genotyping methods such as SNP-Analysis, MLVA 31 Marker and SNR-Analysis to determine the diversity of B. anthracis strains present in Namibia. Based on a total of 44 Namibian genotypes (GTs) ArcGIS was used for the cartography of isolates and the spatial, temporal and spatio-temporal cluster analysis for the GTs found in ENP. A statistical approach was used to compare results with previous studies carried out in other National Parks. The goal of this dissertation was to show the advantages of molecular fingerprinting methods over routine bacteriology of B. anthracis as well as their use in establishing disease patterns. Sample material included historic and recent carcass swabs of wild and domestic animals originating from Namibia as well as soil samples obtained from different habitats within ENP. SNP-analysis revealed four distinct phylogenetic groups that imply four different entries of B. anthracis strains into Namibia (Beyer et al., 2012). Analysis of 525 isolates using MLVA 31 Marker determined that 44 distinct GTs are found in Namibia, of which 24 have been shown to be exclusively present in ENP. Moreover, 268 isolates of five selected GTs for SNR analysis represent a total of 53 SNR-types. It is further eminent that certain B. anthracis GTs predominantly did affect selected species such as zebra and springbok and specific regions within the Park. Spatial and species-specific distribution of GTs was attributed to certain migration patterns of wildlife species such as zebra and elephants in the ENP and showed correlation with specific soil types and vegetation zones. Comparisons of the cluster of diagnostic findings of B. anthracis with GTs did, contrary to expectation, show a much smaller cluster for the most dominant GT (53% of ENP isolates) than the second most dominant (22% of ENP- isolates). GT clustering failed to reveal disease pattern not already apparent from clustering of findings of conventional diagnostics. Beyer et al. (2012) postulated Anthrax outbreaks in ENP as recurrent or ongoing events lasting for different time periods. While the predominant number of historic cases in wildlife species in ENP was reported for the rainy season, continuous surveillance during rainy and dry season in ENP did not show significant seasonal variation in disease cases. Results previously obtained by Lindeque (1991) regarding the seasonal increase of Anthrax cases could only be confirmed partially. Studies of the fate of B. anthracis in different habitats within ENP seem to support the “incubator” theory, except for one of the analyzed categories. Further data are necessary to confirm this trend. The herein presented data seem to be reflective of a seasonal dependent infection risk. Although a higher proportion of samples tested positive in the dry season compared to the rainy season, the B. anthracis concentration of positive cases from the latter season were higher, indicative for a higher risk for lethal infections. The observed overlapping pattern of GT clusters coincided with the occurrence of multiple strain infections. Often different GTs were found in the same habitat and were isolated both from wildlife species and water holes, indicating these water places to be potential sources of infections

Lethal exposure: An integrated approach to pathogen transmission via environmental reservoirs.

To mitigate the effects of zoonotic diseases on human and animal populations, it is critical to understand what factors alter transmission dynamics. Here we assess the risk of exposure to lethal concentrations of the anthrax bacterium, Bacillus anthracis, for grazing animals in a natural system over time through different transmission mechanisms. We follow pathogen concentrations at anthrax carcass sites and waterholes for five years and estimate infection risk as a function of grass, soil or water intake, age of carcass sites, and the exposure required for a lethal infection. Grazing, not drinking, seems the dominant transmission route, and transmission is more probable from grazing at carcass sites 1-2 years of age. Unlike most studies of virulent pathogens that are conducted under controlled conditions for extrapolation to real situations, we evaluate exposure risk under field conditions to estimate the probability of a lethal dose, showing that not all reservoirs with detectable pathogens are significant transmission pathways

Percentage of isolates of the most prevalent GTs in the ENP per year.

<p>Numbers are given for years 2005–2010 when most of the samples were collected. Years 1983, 1987 and 1988–2002, including sampling gaps in 1990, 1993, 1996–99, and 2001, are pooled into the first data point. Total sample sizes are shown below x-axis.</p

Genetic distance as a function of temporal distance and host species similarity.

<p>Isolates of <i>Bacillus anthracis</i> collected from the ENP during the period 1983–2010 are included.</p

Median probabilities and 95% credible intervals for genotypes sampled from the Etosha National Park.

<p>GTs occurring ≤5 times are pooled into the “Other” category.</p

Distribution of genotypes isolated by year.

<p>The total number of isolates per year and per GT. GTs 2, 3, 6, 9, 18, and 22 (highlighted in grey) were found both within and outside the ENP.</p

Confirmed cases of anthrax in the four most commonly affected species in the ENP.

<p>The period between 1988–2010 is shown. Isolates prior to 1988 have been excluded from this figure, having been collected during only short field studies in the Park. The insert indicates the mean monthly rain fall (in l/m²) in the Okaukuejo region, in 1983–2010.</p