Early divergent strains of Yersinia pestis in Eurasia 5,000 years ago.

The bacteria Yersinia pestis is the etiological agent of plague and has caused human pandemics with millions of deaths in historic times. How and when it originated remains contentious. Here, we report the oldest direct evidence of Yersinia pestis identified by ancient DNA in human teeth from Asia and Europe dating from 2,800 to 5,000 years ago. By sequencing the genomes, we find that these ancient plague strains are basal to all known Yersinia pestis. We find the origins of the Yersinia pestis lineage to be at least two times older than previous estimates. We also identify a temporal sequence of genetic changes that lead to increased virulence and the emergence of the bubonic plague. Our results show that plague infection was endemic in the human populations of Eurasia at least 3,000 years before any historical recordings of pandemics.

Gastrointestinal helminths of gray wolves (Canis lupus lupus) from Sweden.

As the Scandinavian wolf population is limited in size, it is only rarely subject to systematic studies on its disease biology, especially gastrointestinal parasites. Therefore, this study aims to describe the prevalence of gastrointestinal helminths of gray wolves hunted on a limited license as a part of a wildlife management program. Helminths of 20 wolves were examined post mortem by macroscopy and coprology. Intestinal worms of five species were recovered from 18 wolves (90%): Uncinaria stenocephala (90%), Taenia spp. (45%), Alaria alata (25%), and Mesocestoides spp. (5%). Of the taeniid specimens typed by multiplex PCR and sequencing of the cox1 gene, 25% belonged to Taenia hydatigena and 25% to Taenia krabbei. The overall species diversity was low compared to findings from wolves of the northern hemisphere. Fecal eggs of Eucoleus boehmi were detected in 12 wolves (60%). Fecal metastrongylid larvae were found in seven individuals (39%), but PCR analyses specific for Angiostrongylus vasorum were negative. The wolves were in good body condition suggesting that the parasite infestation had no negative impact on the general health of the examined wolves. Although some of the recovered parasite species have zoonotic or veterinary impact, it is not likely that the spare wolf population pose substantial threat to human or veterinary health.

Echinococcus multilocularis infection in the field vole (Microtus agrestis): an ecological model for studies on transmission dynamics.

We propose a model involving the oral inoculation of Echinococcus multilocularis eggs in a vole species and examine the infection dynamics in a dose-response experiment. Defined doses, 100 (n = 8), 500 (n = 5) and 1000 (n = 5) of E. multilocularis eggs were used to inoculate Microtus agrestis. Four female C57BL/6j mice were inoculated with 1000 eggs as positive controls. The groups inoculated with 100 and 500 eggs exhibited significantly higher lesion numbers, and relatively smaller lesion size was observed in the 1000 dose group. Undetectable abortive lesions may be responsible for some form of resource limitation early in the infection, resulting in lower lesion counts and size in the 1000 dose group. The C57BL/6j mice exhibited significantly fewer lesions than M. agrestis. The feasibility of measuring corticosterone (which has been shown to downregulate Th1 cytokines) in rodent hair and tumour necrosis factor (TNF) production in spleen cells was demonstrated by a positive correlation between corticosterone levels and higher lesion counts and TNF production in C57BL/6j, respectively. These results suggest that M. agrestis is more prone to a Th2 immune response than C57BL/6j, which is associated with E. multilocularis susceptibility and may explain why the parasite develops more slowly in murine models. This is the first data to suggest that M. agrestis is capable of supporting E. multilocularis transmission and thus may be suited as a model to describe the infection dynamics in an intermediate host that affects transmission under natural conditions.

Detection of zoonotic Cryptosporidium spp. in small wild rodents using amplicon-based next-generation sequencing.

Rodents may serve as reservoirs of zoonotic species of Cryptosporidium; however, data from molecular surveys in support of this hypothesis are still scarce. In this study, we screened faeces and rectal content from murid and cricetid rodents (N = 58) caught around three farms in Zealand, Denmark, for Cryptosporidium spp. by amplicon-based next-generation sequencing (NGS) of ribosomal genes. Selected samples were further examined using nested conventional PCR targeting SSU rRNA, gp60, and actin genes. Cryptosporidium-specific DNA was identified in 40/58 (69%) samples, and in 12 (30%) of the 40 positive animals, mixed cryptosporidial infections were observed. Cryptosporidium ditrichi was the species most commonly identified, found in 28 (48%) of the animals. Cryptosporidium parvum was identified in 4 (7%) of the animals, all of which were co-infected with C. ditrichi. The present study is the first to utilize NGS-based screening for Cryptosporidium species in wild rodents. Moreover, it is the first study to provide molecular data on Cryptosporidium in rodents sampled in Denmark and to detect DNA of C. ditrichi in Mus musculus, Myodes glareolus, and Microtus agrestis. The NGS approach was successfully applied to yield new knowledge, and the results showed that zoonotic species of Cryptosporidium are common in murid and cricetid rodents in Zealand, Denmark.

Population genomics of ancient and modern Trichuris trichiura.

The neglected tropical disease trichuriasis is caused by the whipworm Trichuris trichiura, a soil-transmitted helminth that has infected humans for millennia. Today, T. trichiura infects as many as 500 million people, predominantly in communities with poor sanitary infrastructure enabling sustained faecal-oral transmission. Using whole-genome sequencing of geographically distributed worms collected from human and other primate hosts, together with ancient samples preserved in archaeologically-defined latrines and deposits dated up to one thousand years old, we present the first population genomics study of T. trichiura. We describe the continent-scale genetic structure between whipworms infecting humans and baboons relative to those infecting other primates. Admixture and population demographic analyses support a stepwise distribution of genetic variation that is highest in Uganda, consistent with an African origin and subsequent translocation with human migration. Finally, genome-wide analyses between human samples and between human and non-human primate samples reveal local regions of genetic differentiation between geographically distinct populations. These data provide insight into zoonotic reservoirs of human-infective T. trichiura and will support future efforts toward the implementation of genomic epidemiology of this globally important helminth.

Parasitic infections and resource economy of Danish Iron Age settlement through ancient DNA sequencing.

In this study, we screen archaeological soil samples by microscopy and analyse the samples by next generation sequencing to obtain results with parasites at species level and untargeted findings of plant and animal DNA. Three separate sediment layers of an ancient man-made pond in Hoby, Denmark, ranging from 100 BC to 200 AD, were analysed by microscopy for presence of intestinal worm eggs and DNA analysis were performed to identify intestinal worms and dietary components. Ancient DNA of parasites, domestic animals and edible plants revealed a change in use of the pond over time reflecting the household practice in the adjacent Iron Age settlement. The most abundant parasite found belonged to the Ascaris genus, which was not possible to type at species level. For all sediment layers the presence of eggs of the human whipworm Trichuris trichiura and the beef tapeworm Taenia saginata suggests continuous disposal of human faeces in the pond. Moreover, the continuous findings of T. saginata further imply beef consumption and may suggest that cattle were living in the immediate surrounding of the site throughout the period. Findings of additional host-specific parasites suggest fluctuating presence of other domestic animals over time: Trichuris suis (pig), Parascaris univalens (horse), Taenia hydatigena (dog and sheep). Likewise, alternating occurrence of aDNA of edible plants may suggest changes in agricultural practices. Moreover, the composition of aDNA of parasites, plants and vertebrates suggests a significant change in the use of the ancient pond over a period of three centuries.

Ancient DNA from latrines in Northern Europe and the Middle East (500 BC-1700 AD) reveals past parasites and diet.

High-resolution insight into parasitic infections and diet of past populations in Northern Europe and the Middle East (500 BC- 1700 AD) was obtained by pre-concentration of parasite eggs from ancient latrines and deposits followed by shotgun sequencing of DNA. Complementary profiling of parasite, vertebrate and plant DNA proved highly informative in the study of ancient health, human-animal interactions as well as animal and plant dietary components. Most prominent were finding of soil-borne parasites transmitted directly between humans, but also meat-borne parasites that require consumption of raw or undercooked fish and pork. The detection of parasites for which sheep, horse, dog, pig, and rodents serves as definitive hosts are clear markers of domestic and synanthropic animals living in closer proximity of the respective sites. Finally, the reconstruction of full mitochondrial parasite genomes from whipworm (Ascaris lumbricoides) and roundworm species (Trichuris trichiura and Trichuris muris) and estimates of haplotype frequencies elucidates the genetic diversity and provides insights into epidemiology and parasite biology.

Peroral Echinococcus multilocularis egg inoculation in Myodes glareolus, Mesocricetus auratus and Mus musculus (CD-1 IGS and C57BL/6j).

Echinococcus multilocularis transmission predominantly occurs in Europe between the red fox (Vulpes vulpes) and various species of rodent intermediate hosts. We infected 3 species of rodent, Myodes glareolus (n = 47), Mesocricetus auratus (n = 11) and outbred Mus musculus (CD-1 IGS) (n = 9) with an E. multilocularis egg suspension that contained 100 eggs with viable oncospheres and performed post mortem examination 6, 8 (M. glareolus) and 10 weeks post inoculation (wpi). C57BL/6j mice (n = 4) were used as positive controls as they have been shown to exhibit macroscopic liver lesions 4 wpi. To the best of our knowledge, this is the first study to experimentally assess susceptibility in the ostensibly competent host M. glareolus. Lesions were only detected in 2 of 47 M. glareolus (4.3%) at 8 and 10 wpi and although both contained protoscolices (1675 at 8 wpi and 88 at 12 wpi) the low percentage of infected animals brings into question their role as transmitters of the parasite. Significant differences were observed between inbred and outbred mice with E. multilocularis infection in the former demonstrating increased establishment (p ≤ 0.0001) and growth (p ≤ 0.0001). No lesions were found in all 11 M. auratus.

Establishment and development of Echinococcus multilocularis metacestodes in the common vole (Microtus arvalis) after oral inoculation with parasite eggs.

Transmission of the zoonotic tapeworm, Echinococcus multilocularis mainly occurs between the red fox (Vulpes vulpes) and various species of vole. Microtus arvalis is considered one of the key intermediate hosts in Europe. We infected 21 M. arvalis aged 35 days (n=2), 56 days (n=6), 84 days (n=4) and 263 days (n=9) with 100 E. multilocularis eggs. Four voles aged 263 days were euthanized at 6 weeks post inoculation (wpi) with the remainder euthanized 10 wpi for analysis of metacestode growth and protoscolex development. Eight C57BL/6j mice (age 35-231 days) were included as controls for egg viability (they have been shown to exhibit visible infection after 4 wpi) and dissected at 6 (n=2) and 10 (n=6) wpi. M. arvalis had significantly higher metacestode establishment (p=0.008) 6 wpi with 27.5±6.63S.D. compared to C57BL/6j with 15.5±0.71S.D. Multivesiculation precluded enumeration at 10 wpi in M. arvalis. No protoscolices were found in metacestodes in M. arvalis 6 wpi or C57BL/6j at any time point but were found in all infected voles 10 wpi (48,056±52,574 S.D.). It has been reported that glucocorticoid (GC) profile can affect E. multilocularis establishment. This was assessed by measuring corticosterone in rodent hair to determine if parasite establishment or fertility was related to this stress hormone. No significant differences were found. Data presented here provides, for the first time, a protoscolex development window in this species that has the potential to shed light on the epizootiology of this parasite.

DNA typing of ancient parasite eggs from environmental samples identifies human and animal worm infections in Viking-age settlement.

Ancient parasite eggs were recovered from environmental samples collected at a Viking-age settlement in Viborg, Denmark, dated 1018-1030 A.D. Morphological examination identified Ascaris sp., Trichuris sp., and Fasciola sp. eggs, but size and shape did not allow species identification. By carefully selecting genetic markers, PCR amplification and sequencing of ancient DNA (aDNA) isolates resulted in identification of: the human whipworm, Trichuris trichiura , using SSUrRNA sequence homology; Ascaris sp. with 100% homology to cox1 haplotype 07; and Fasciola hepatica using ITS1 sequence homology. The identification of T. trichiura eggs indicates that human fecal material is present and, hence, that the Ascaris sp. haplotype 07 was most likely a human variant in Viking-age Denmark. The location of the F. hepatica finding suggests that sheep or cattle are the most likely hosts. Further, we sequenced the Ascaris sp. 18S rRNA gene in recent isolates from humans and pigs of global distribution and show that this is not a suited marker for species-specific identification. Finally, we discuss ancient parasitism in Denmark and the implementation of aDNA analysis methods in paleoparasitological studies. We argue that when employing species-specific identification, soil samples offer excellent opportunities for studies of human parasite infections and of human and animal interactions of the past.

Predictors of Echinococcus multilocularis Prevalence in Definitive and Intermediate Hosts: A Meta-Analysis Approach.

Echinococcus multilocularis (EM) is a pathogenic and potentially fatal cestode causing human alveolar echinococcosis (AE). A meta-analysis was conducted using a generalized estimation equation approach (GEE) to assess the effect of taxonomic, environmental, and diagnostic variables on EM prevalence in different hosts. Red foxes ( Vulpes vulpes ) had significantly higher prevalence of EM than domestic dogs ( Canis lupus familiaris), with the diagnostic method playing an important factor in assessing prevalence. For intermediate hosts genera was significantly associated with EM prevalence, although there was some indication of publication bias in this dataset. This study also highlights the possible importance of temperature and precipitation to EM transmission. This implies the possibility of a changing climate affecting the future distribution of the parasite.

First report of Eucoleus boehmi in red foxes (Vulpis vulpis) in Denmark, based on coprological examination.

Red foxes can be infected with diverse range of parasite species that can be transmitted to humans and pet animals, and the differential diagnosis is essential for veterinary and zoonotic risk assessment. In the present study, faecal flotation and microscopy of parasite eggs was done on 31 foxes originating from two distant localities in Denmark, the city of Copenhagen in the north east part of the island Zealand and from the southern part of the peninsular Jutland. In total, eggs of Eucoleus boehmi were recovered from a surprisingly high number of foxes (n = 22 samples; 71%). The findings represent the first observations of E. boehmi in Denmark, which is likely an overlooked infection of the upper respiratory tract of red foxes, which can be also found in other canids. Several morphological features of the eggs of E. boehmi can be used to distinguish it from other the closely related trichuroid eggs. Detecting cardiopulmonary parasites by faecal examination can be indicative of the presence of cardiopulmonary parasites based on the more sensitive post mortem analysis.

Warble infestations by Hypoderma tarandi (Diptera; Oestridae) recorded for the first time in West Greenland muskoxen.

In the northern hemisphere, Caribou (Rangifer spp.) populations are known to be infested with the skin-penetrating ectoparasite, Hypoderma tarandi (Diptera; Oestridae). Although regarded as host specific, H. tarandi has been reported from other species, and has become of increasing concern as a zoonosis infecting humans. In February 2012, concurrent with the hunting of muskoxen, we examined carcasses for muscle and tissue parasites, and recorded warble larvae infestations. DNA extracted from samples of larvae was amplified targeting 579 bp of the COI gene, and subsequently sequenced, to be confirmed as H. tarandi. Infestation by oestrid flies has not previously been reported in muskoxen in West Greenland.

First record of Taenia ovis krabbei muscle cysts in muskoxen from Greenland.

A first record of Taenia ovis krabbei muscle cysts in a muskoxen (Ovibos moschatus) from the Kangerlussuaq population in West Greenland suggests that introduced muskoxen now contributes to the transmission of this parasite in addition to previous observations from caribou (Rangifer tarandus). Muskoxen and caribou are the only wild ungulates in Greenland.