Spatial heterogeneity and temporal variations in Echinococcus multilocularis infections in wild hosts in a North American urban setting

Echinococcus multilocularis, the causative agent of human alveolar echinococcosis, has the potential to circulate in urban areas where wild host populations and humans coexist. The spatial and temporal distribution of infection in wild hosts locally affects the risk of transmission to humans. We investigated the spatial and temporal patterns of E. multilocularis infection in coyotes and rodent intermediate hosts within the city of Calgary, Canada, and the association between spatial variations in coyote infection and the relative composition of small mammal assemblages. Infection by E. multilocularis was examined in small mammals and coyote faeces collected monthly in five city parks from June 2012 to June 2013. Coyote faeces were analysed using a ZnCl₂ centrifugation and sedimentation protocol. Infection in intermediate hosts was assessed through lethal trapping and post-mortem analysis. Parasite eggs and metacestodes were morphologically identified and molecularly confirmed through species-specific PCR assays. Of 982 small mammals captured, infection was detected in 2/305 (0.66%) deer mice (Peromyscus maniculatus), 2/267 (0.75%) meadow voles (Microtus pennsylvanicus), and 1/71 (1.41%) southern red backed voles (Myodes gapperi). Overall faecal prevalence in coyotes was 21.42% (n = 385) and varied across sites, ranging from 5.34% to 61.48%. Differences in coyote faecal prevalence across sites were consistent with local variations in the relative abundance of intermediate hosts within the small mammal assemblages. Infections peaked in intermediate hosts during autumn (0.68%) and winter (3.33%), and in coyotes during spring (43.47%). Peaks of infections in coyote faeces up to 83.8% in autumn were detected in a hyper-endemic area. To the best of our knowledge, our findings represent the first evidence of a sylvatic life-cycle of E. multilocularis in a North American urban setting, and provide new insights into the complexity of the parasite transmission ecology.     

A new intermediate host for Echinococcus multilocularis: The southern red-backed vole (Myodes gapperi) in urban landscape in Calgary,Canada

Human Alveolar Echinococcosis (HAE) is a potentially fatal parasitic disease caused by Echinococcus multilocularis, a cestode characterized by a sylvatic life-cycle involving several species of rodents and lagomorphs as intermediate hosts and canids as definitive hosts. Despite the wide distribution of the parasite in North America, the number of competent intermediate host species identified to date is still relatively small, and mainly includes the northern vole (Microtus oeconomus), brown lemming (Lemmus sibiricus), northern red-backed vole (Myodes rutilus), deer mouse (Peromyscus maniculatus) and meadow vole (Microtus pennsylvanicus).By monitoring the infections in rodents in the city of Calgary (Alberta, Canada), we have detected a case of severe alveolar echinococcosis in a southern red-backed vole (Myodes gapperi), a species never reported before as an intermediate host for this parasite. Observation of protoscolices in the intra-abdominal multilocular cysts indicates that M. gapperi could act as a competent intermediate host for the transmission of E. multilocularis.Since M. gapperi can be found in close proximity to, and within metropolitan areas, this species could play a role in the establishment and maintenance of the sylvatic life-cycle of E. multilocularis in urban landscapes, where the potential for zoonotic transmission is higher. The new intermediate host reported needs to be taken into account in future surveys and transmission models for this parasite.     

Does Removing Coyotes for Livestock Protection Benefit Free-Ranging Ungulates?

Abstract: We studied the effects of coyote (Canis latrans) control for livestock protection on native ungulates during 2003 and 2004 on 7 sites in Utah and Colorado, USA, totaling over 1,900 km². We found no relationships between coyote control variables and offspring/female deer ratios. However, control effort (no. of hr spent aerial gunning for coyotes) and success (no. of coyotes taken) were positively correlated with numbers of mule deer (Odocoileus hemionus) and pronghorn (Antilocapra americana) observed per kilometer of transect. Our results suggest that coyote control for livestock protection may increase densities of mule deer and pronghorn in areas where it is conducted.     

Evaluating the effect of predators on white-tailed deer: Movement and diet of coyotes

Coyotes (Canis latrans) may affect adult and neonate white-tailed deer (Odocoileus virginianus) survival and have been implicated as a contributor to the decline of deer populations. Additionally, coyote diet composition is influenced by prey availability, season, and region. Because coyote movement and diet vary by region, local data are important to understand coyote population dynamics and their impact on prey species. In southeast Minnesota, we investigated the effect of coyotes on white-tailed deer populations by documenting movement rates, distances moved, and habitats searched by coyotes during fawning and nonfawning periods. Additionally, we determined survival, cause-specific mortality, and seasonal diet composition of coyotes. From 2001 to 2003, we captured and radiocollared 30 coyotes. Per-hour rate of movement averaged 0.87 km and was greater (P = 0.046) during the fawning (1.07 km) than the nonfawning period (0.80 km); areas searched were similar (P = 0.175) between seasons. Coyote habitat use differed during both seasons; habitats were not used in proportion to their availability (P < 0.001). Croplands were used more (P < 0.001) than their proportional availability during both seasons. Use of grasslands was greater during the fawning period (P = 0.030), whereas use of cropland was greater in the nonfawning period (P < 0.001). We collected 66 fecal samples during the nonfawning period; coyote diets were primarily composed of Microtus spp. (65.2%), and consumption of deer was 9.1%. During the study, 19 coyotes died; annual survival rate range was 0.33–0.41, which was low compared with other studies. Consumption of deer was low and coyotes searched open areas (i.e., cropland) more than fawning areas with dense cover. These factors in addition to high coyote mortality suggested that coyote predation was not likely limiting white-tailed deer populations in southeast Minnesota. © 2011 The Wildlife Society.     

Can Coyotes Affect Deer Populations in Southeastern North America?

ABSTRACT The coyote (Canis latrans) is a recent addition to the fauna of eastern North America, and in many areas coyote populations have been established for only a decade or two. Although coyotes are known predators of white-tailed deer (Odocoileus virginianus) in their historic range, effects this new predator may have on eastern deer populations have received little attention. We speculated that in the southeastern United States, coyotes may be affecting deer recruitment, and we present 5 lines of evidence that suggest this possibility. First, the statewide deer population in South Carolina has declined coincident with the establishment and increase in the coyote population. Second, data sets from the Savannah River Site (SRS) in South Carolina indicate a new mortality source affecting the deer population concurrent with the increase in coyotes. Third, an index of deer recruitment at SRS declined during the period of increase in coyotes. Fourth, food habits data from SRS indicate that fawns are an important food item for coyotes during summer. Finally, recent research from Alabama documented significant coyote predation on fawns there. Although this evidence does not establish cause and effect between coyotes and observed declines in deer recruitment, we argue that additional research should proactively address this topic in the region. We identified several important questions on the nature of the deer—coyote relationship in the East.     

High endemicity of alveolar echinococcosis in Yili Prefecture,Xinjiang Autonomous Region,the People’s Republic of China: Infection status in different ethnic communities and in small mammals

Alveolar echinococcosis (AE) is a life-threatening disease in humans caused by the larval stage of Echinococcus multilocularis. The tapeworm is transmitted between small mammals and dogs/foxes in the Northern Hemisphere. In this study 286 AE cases were reported from eight counties and one city in Yili Prefecture, Xinjiang Autonomous Region, the People’s Republic of China from 1989 to 2015 with an annual incidence (AI) of 0.41/100,000. Among the patients, 73.08% were diagnosed in the last 11 years. Four counties in the high mountainous areas showed higher AI (0.51–1.22 cases/100,000 residents) than the four counties in low level areas (0.19–0.29/100,000 residents). The AI of AE in Mongolian (2.06/100,000 residents) and Kazak (0.93/100,000 residents) ethnic groups was higher than the incidence in other ethnic groups indicating sheep-farming is a risk for infection given this activity is mainly practiced by these two groups in the prefecture. A total of 1411 small mammals were captured with 9.14% infected with E. multilocularis metacestodes. Microtus obscurus was the dominant species in the mountain pasture areas with 15.01% of the voles infected, whereas Mus musculus and Apodemus sylvaticus were the dominant small mammals in the low altitude areas. Only 0.40% of A. sylvaticus were infected with E. multilocularis. PCR amplification and sequencing analysis of the mitochondrial cox1 gene showed that E. multilocularis DNA sequences from the small mammals were identical to isolates of local human AE cases. The overall results show that Yili Prefecture is a highly endemic area for AE and that the high-altitude pasture areas favorable for M. obscurus may play an important role in its transmission in this region.     

A MEKK1 – JNK mitogen activated kinase (MAPK) cascade module is active in Echinococcus multilocularis stem cells

BackgroundThe metacestode larval stage of the fox-tapeworm Echinococcus multilocularis causes alveolar echinococcosis by tumour-like growth within the liver of the intermediate host. Metacestode growth and development is stimulated by host-derived cytokines such as insulin, fibroblast growth factor, and epidermal growth factor via activation of cognate receptor tyrosine kinases expressed by the parasite. Little is known, however, concerning signal transmission to the parasite nucleus and cross-reaction with other parasite signalling systems.Methodology/Principal findingsUsing bioinformatic approaches, cloning, and yeast two-hybrid analyses we identified a novel mitogen-activated kinase (MAPK) cascade module that consists of E. multilocularis orthologs of the tyrosine kinase receptor interactor Growth factor receptor-bound 2, EmGrb2, the MAPK kinase kinase EmMEKK1, a novel MAPK kinase, EmMKK3, and a close homolog to c-Jun N-terminal kinase (JNK), EmMPK3. Whole mount in situ hybridization analyses indicated that EmMEKK1 and EmMPK3 are both expressed in E. multilocularis germinative (stem) cells but also in differentiated or differentiating cells. Treatment with the known JNK inhibitor SP600125 led to a significantly reduced formation of metacestode vesicles from stem cells and to a specific reduction of proliferating stem cells in mature metacestode vesicles.Conclusions/SignificanceWe provide evidence for the expression of a MEKK1-JNK MAPK cascade module which, in mammals, is crucially involved in stress responses, cytoskeletal rearrangements, and apoptosis, in E. multilocularis stem cells. Inhibitor studies indicate an important role of JNK signalling in E. multilocularis stem cell survival and/or maintenance. Our data are relevant for molecular and cellular studies into crosstalk signalling mechanisms that govern Echinococcus stem cell function and introduce the JNK signalling cascade as a possible target of chemotherapeutics against echinococcosis.     

Population-level response of coyotes to a pulsed resource event

From foraging theory, generalist predators should increase consumption of prey if prey availability increases. Pulsed resource events introduce a large influx of prey to predators that may exhibit a functional response of increased consumption rate on, or specialization to, this abundant food resource. We predicted that coyotes (Canis latrans) would respond functionally to numerical increases of neonate white-tailed deer (Odocoileus virginianus) during the pulsed resource event of parturition. We used howl surveys and deer camera surveys with occupancy modeling to estimate densities for coyotes, adult deer, and fawns, respectively, in Upper Peninsula of Michigan, USA, 2009–2011. We estimated biomass of adult and fawn deer consumed by coyotes during 2 periods [fawn limited mobility period (LMP) and social mobility period (SMP)] in May–August each year. Coyote densities were 0.32 and 0.37/km² for 2010–2011, respectively. Adult deer densities (3.7–3.9/km²) and fawn densities (0.6–1.3/km²) were similar across years. Overall, fawn hair occurrence in coyote scats was 2.3 times greater in LMP than SMP. Estimated consumption of fawns between periods (n = 157–880) by coyotes varied, suggesting a functional response, with increasing consumption of fawns relative to their availability. Coyotes, on average, consumed 2.2 times greater biomass of fawns than adults across years, and consumed 1.5 times greater fawn biomass, on average, during LMP than SMP. We suggest that consumption rates of coyotes is associated positively with increases in fawn density, and fawn consumption by coyotes follows predictions of optimal foraging theory during this pulsed resource event.     

Effects of large-scale removal of coyotes on pronghorn and mule deer productivity and abundance

We tested the hypothesis that predation by coyotes (Canis latrans) impacts pronghorn (Antilocapra americana) and mule deer (Odocoileus hemionus) populations. We did so by examining the effects of coyote removal on pronghorn and mule deer populations within 12 large areas (>10,500 km²) located in Wyoming and Utah during 2007 and 2008. Pronghorn productivity (fawn to adult female ratio) and abundance were positively correlated with the number of coyotes removed and removal effort (hours spent hunting coyotes from aircraft) although the correlation between pronghorn productivity and removal effort was not statistically significant (P = 0.08). Mule deer productivity and abundance were not correlated with either the number of coyotes removed or removal effort. Coyote removal conducted during the winter and spring provided greater benefit than removals conducted during the prior fall or summer. Our results suggest that coyote removal conducted over large areas increases fawn survival and abundance of pronghorn but not mule deer. © 2011 The Wildlife Society.     

Comparison of Mexican Wolf and Coyote Diets in Arizona and New Mexico

ABSTRACT Interactions between wolves (Canis lupus) and coyotes (C. latrans) can have significant impacts on their distribution and abundance. We compared diets of recently translocated Mexican wolves (C. l. baileyi) with diets of resident coyotes in Arizona and New Mexico, USA. We systematically collected scats during 2000 and 2001. Coyote diet was composed mostly of mammalian species, followed by vegetation and insects. Elk (Cervus elaphus) was the most common item in coyote scats. Mexican wolf diet had a higher proportion of large mammals and fewer small mammals than coyote diet; however, elk was also the most common food item in Mexican wolf scats. Our results suggest that Mexican wolf diet was more similar to coyote diet than previously reported, but coyotes had more seasonal variation. Considering results in other areas, we expect that Mexican wolves will have a negative impact on coyotes through direct mortality and possibly competition. Reintroduction of Mexican wolves may have great impacts on communities by changing relationships among other predators and their prey.     

Coyotes as Sentinels for Monitoring Bovine Tuberculosis Prevalence in White-Tailed Deer

Abstract: Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB), is endemic in free-ranging white-tailed deer (Odocoileus virginianus) in 5 counties (Alcona, Alpena, Montmorency, Oscoda, and Presque Isle) in the northeastern Lower Peninsula of Michigan, USA. The presence of a wildlife reservoir of tuberculosis in Michigan and the incidence of bTB in cattle (Bos taurus) resulted in Michigan losing its bTB accredited-free status. Subsequent wildlife surveillance programs identified relatively high disease prevalence in coyotes (Canis latrans), generating interest in their potential to serve as a sentinel species to detect bTB prevalence in white-tailed deer. Our goal was to develop an empirical basis for generating hypotheses about the spatial epidemiology of bTB infection in coyotes for future surveillance, management, and modeling efforts. Though variation in coyote home-range size may confound attempts to spatially correlate the incidence of disease in the sentinel and host species at a fine scale, overlap zones (OZs) between adjacent coyote home ranges may be the appropriate sample unit for spatially correlating disease prevalence in coyotes and white-tailed deer. Because overlapping home ranges are generally configured around resource rich (e.g., small mammals and white-tailed deer) timber management patches, the OZ concentrates spatial interaction between adjacent groups in a relatively small area. Furthermore, there is a direct relationship between interaction probabilities and the spatial dispersion of those patches. The latter finding provides a useful metric to incorporate into future efforts to develop spatially explicit models of bTB dynamics. Modeling efforts can then be used as a foundation to predict the epidemiological ramifications of alterations in intensively managed forested landscapes. (JOURNAL OF WILDLIFE MANAGEMENT 71(5):1545-1554; 2007)     

Echinococcosis in China,a Review of the Epidemiology of <Emphasis Type="Italic">Echinococcus</Emphasis> spp.

Cystic echinococcosis (CE) and alveolar echinococcosis (AE) are highly significant infectious diseases occurring worldwide and caused by metacestodes of tapeworms Echinococcus granulosus and E. multilocularis, respectively. Both human CE and AE have highest prevalence rates in western and northwestern China. Livestock is the main intermediate host of E. granulosus, and wild small mammal are the main intermediate hosts of E. multilocularis. Since they range freely in pastoral areas, prey on wild small mammals and offal of livestock after slaughter, and have close relationships with humans, domestic dogs are the most important definitive host of both Echinococcus spp. with the highest risk of transmitting CE and AE to humans. Pastoralism is the occupation with the highest risk of being infected with the both kinds of echinococcosis due to the proximity of livestock, dogs, and wildlife host species. In this review, we summarize the epidemiology of human echinococcosis, the situation of parasite transmission in animal hosts, and possible transmission patterns in China. In addition, human activities and their potential influence on the transmission of echinococcosis are also discussed.     

Territory fidelity, space use, and survival rates of wild coyotes following surgical sterilization

Sterilization of wild canids is being used experimentally in many management applications. Few studies have clearly demonstrated vasectomized and tubal-ligated canids will retain pair-bonding and territorial behaviors. We tested whether territory fidelity, space use, and survival rates of surgically sterilized coyote (Canis latrans) packs were different from sham-operated coyote packs. We captured and radio-collared 30 coyotes in December 2006. Sixteen of these animals were sterilized via vasectomy or tubal ligation, and 14 were given sham-surgeries (i.e., remained intact). We monitored these animals using telemetry and visual observations through 2 breeding seasons and 1 pup-rearing season from December 2006 to March 2008. Mean pack size was not significantly different between sterile and intact coyote packs. We found no difference in home range size between sterile and intact coyotes. We found differences in home range and core area overlap between sterile and intact coyote packs in some seasons; however, this difference may have existed prior to sterilization. Home range fidelity was not significantly different between sterile and intact coyotes. All coyotes had higher residency rates during the breeding season, with no differences between sterile and intact coyotes. Survival rates were correlated with biological season, but there were no differences in survival rates between sterile and intact coyotes. We concluded that surgical sterilization of coyotes did not affect territory fidelity, survival rates, or home range maintenance.     

Deep amplicon sequencing highlights low intra-host genetic variability of Echinococcus multilocularis and high prevalence of the European-type haplotypes in coyotes and red foxes in Alberta,Canada

Echinococcus multilocularis (Em) is a zoonotic parasite considered a global emergent pathogen. Recent findings indicate that the parasite is expanding its range in North America and that European-type haplotypes are circulating in western Canada. However, genetic analyses are usually conducted only on a few parasites out of thousands of individuals within each definitive host, likely underestimating the prevalence of less common haplotypes. Moreover, mixed infections with several mtDNA haplotypes in the same host have been reported, but their relative abundance within the host was never estimated. We aimed to 1) estimate the frequency of co-infections of different Em haplotypes in coyotes (Canis latrans) and red foxes (Vulpes vulpes) from western Canada and their relative abundance within the definitive hosts, 2) detect less prevalent haplotypes by sampling a larger proportion of the parasite subpopulation per host, and 3) investigate differences in the distribution of Em haplotypes in these main definitive hosts; foxes and coyotes. We extracted DNA from ~10% of the worm subpopulation per host (20 foxes and 47 coyotes) and used deep amplicon sequencing (NGS technology) on four loci, targeting the most polymorphic regions from the mitochondrial genes cox1 (814 bp), nad1 (344 bp), and cob (387 bp). We detected the presence of mixed infections with multiple Em haplotypes and with different Echinococcus species including Em and E. granulosus s.l. genotypes G8/G10, low intraspecific diversity of Em, and a higher abundance of the European-type haplotypes in both hosts. Our results suggest a population expansion of the European over the North American strain in Alberta and a limited distribution of some European-type haplotypes. Our findings indicate that deep amplicon sequencing represents a valuable tool to characterize Em in multiple hosts, to assess the current distribution and possible origins of the European strain in North America. The potential use of next-generation sequencing technologies is particularly important to understand the patterns of geographic expansion of this parasite.     

Shifts from native to invasive small mammals across gradients from tropical forest to urban habitat in Borneo

Urbanization has paved the way for the spread of commensal rodents at global scale. However, it is largely unknown how these species use tropical anthropogenic landscapes originally covered with forests and inhabited by diverse small mammal assemblages. We surveyed non-flying small mammals in various urban and suburban habitat types and adjacent forest in the tropical town of Kota Kinabalu in Borneo. We used occupancy and polynomial regression models to determine variation in species occurrences along gradients of land-use intensity. Müller’s sundamys (Sundamys muelleri) was the only native small mammal species found in urban and suburban landscapes with a continuous decrease in occurrence probability from forests to urban habitats. The invasive Asian black rat (Rattus rattus species complex) and the invasive Asian house shrew (Suncus murinus) had the highest occurrence probabilities in habitats of intermediate land-use intensity, but Asian black rats are also likely to occasionally invade forested habitats and occupied urban habitats in sympatry with the Norway rat (Rattus norvegicus). In urban and suburban habitats, fallow land possibly favoured the occurrence of S. muelleri and S. murinus. Other native small mammal species (Muridae, Sciuridae, Tupaiidae) were found only in forested areas. Our study shows that native small mammals found in forest are largely replaced by invasive species in urban and suburban habitats. Due to their occurrence in habitats of various land use intensities, S. muelleri and R. rattus comprise central links between forest wildlife and urban species, an association that is important to consider in studies of parasite and disease transmission dynamics.     

Urban coyotes select cryptic den sites near human development where conflict rates increase

The establishment of coyote (Canis latrans) populations in urban areas across North America has been accompanied by increased rates of human–coyote conflict. One factor thought to promote physical conflict between coyotes and people or pets is the presence of coyote pups near natal dens; however, this idea has not been tested, and no multivariate study of den selection within cities has occurred. Our objectives were to conduct a multivariate analysis of third- (i.e., home range) and fourth-order (i.e., den sites) habitat selection at dens and determine whether proximity to dens is associated with reports of physical conflict with coyotes. We found 120 dens by following coyote trails using snow tracking within urban green spaces that comprise presumed high-quality habitat for coyotes in Edmonton, Alberta, Canada. We used resource selection functions to assess habitat selection for dens, testing variables related to land cover and anthropogenic features at the third order, and testing microsite habitat features via paired sites at the fourth order. We defined conflict encounters from comments in a community reporting database and used general linear models to assess their spatial proximity to the nearest den and prevalence during the pup-rearing period compared to the rest of the year. Habitat selection was strongest at the fourth order, wherein coyotes selected for abundant hiding cover, steep slopes, and eastern exposure. The prevalence of physical conflict with coyotes increased during the pup-rearing period. Conflict also increased near known dens as an overall effect and when reports occurred outside of naturalized urban areas. These results suggest that coyotes in Edmonton den in green spaces near human development in microsites that minimize detection by people via steep slopes and dense vegetation. We suggest urban wildlife managers increase public safety education about recognition of coyote denning habitat and coyote defensive behaviors, especially outside of naturalized urban areas, because of the observed increase in physical conflict near dens.     

Predation by coyotes on white-tailed deer neonates in South Carolina

Coyotes (Canis latrans) are novel predators throughout the southeastern United States and their depredation of white-tailed deer (Odocoileus virginianus) neonates may explain observed declines in some deer populations in the region, but direct evidence for such a relationship is lacking. Our objective was to quantify neonate survival rates and causes of mortality at the United States Department of Energy's Savannah River Site (SRS), South Carolina to directly evaluate degree of predation in this deer population. From 2006 to 2009, we radio-monitored 91 neonates captured with the aid of vaginal implant transmitters in pregnant adult females and opportunistic searches. Overall Kaplan–Meier survival rate to 16 weeks of age was 0.230 (95% CI = 0.155–0.328), and it varied little among years. Our best-fitting model estimated survival at 0.220 (95% CI = 0.144–0.320). This model included a quadratic time trend variable (lowest survival rate during the first week of life and increasing to near 1.000 around week 10), and Julian date of birth (survival probability declining as date of birth increased). Predation by coyotes was the most frequent cause of death among the 70 monitored neonates that died, definitively accounting for 37% of all mortalities and potentially accounting for as much as 80% when also including probable coyote predation. Predation by bobcats (Felis rufus) accounted for 7% (definitive) to 9% (including probable bobcat predation) of mortalities. The level of coyote-induced mortality we observed is consistent with the low recruitment rates exhibited in the SRS deer population since establishment of coyotes at the site. If representative of recruitment rates across South Carolina, current harvest levels appear unsustainable. This understanding is consistent with the recent declining trend in the statewide deer population. The effects of coyote predation on recruitment should be considered when setting harvest goals, regardless of whether local deer population size is currently above or below desired levels, because coyotes can substantially reduce fawn recruitment. Published 2012. This article is a U.S. Government work and is in the public domain in the USA.     

Establishing and evaluation of a polymerase chain reaction for the detection of Echinococcus multilocularis in human tissue

BackgroundAlveolar echinococcosis (AE) is caused by metacestode larva of the tapeworm Echinococcus multilocularis. AE diagnostics currently rely on imaging techniques supported by serology, but unequivocal detection of AE is difficult. Although polymerase chain reaction (PCR)-based methods to detect tapeworm DNA in biopsies have been suggested for several species, no validated protocol adhering to accepted guidelines has so far been presented for AE diagnostics. We herein established a PCR protocol for metacestode biopsies and technically evaluated the method using isolated parasite DNA and cells, biopsies of clinically relevant material, and formalin fixed paraffin-embedded (FFPE) human tissue blocks. We compared the results with an immunochemical (IHC) approach using the monoclonal antibody Em2G11 specific for the antigen Em2 of E. mulitlocularis.Methodology/Principal findingsBased on tapeworm 12S rDNA sequences we established and validated a PCR protocol for robust detection of as little as 50 parasite cells per specimen and report 127 cases of positive identification of Echinococcus species in samples from humans and animals. For further validation, we analyzed 45 liver, heart, brain, and soft tissue samples as well as cytological probes of aspirates of FFPE-material from 18 patients with clinically confirmed AE. Of each patient we analyzed (i) fully viable lesions with laminated layer; (ii) tissue with mAbEm2G11-positive small particles of E. multilocularis (spems); (iii) mAbEm2G11-negative tissue adjacent to the main lesion; and (iv) lymph node tissue with mAbEm2G11-positive spems. To identify the areas for the PCR-based approach, we performed IHC-staining with the monoclonal antibody Em2G11. Micro-dissected tissue of these areas was then used for PCR-analysis. 9 of 15 analyzed samples with viable E. multilocularis lesions with laminated layer were positive by PCR. Of this group, all samples preserved for less than 6 years (6/6) were tested positive. 11 of 15 samples of spems and 7 of 9 samples of the control group mAbEm2G11-negative tissue were negative by PCR. We further show that all probes from lymph nodes with spems are PCR negative.Conclusions/SignificanceWe present a sensitive PCR method for the detection of E. multilocularis in human tissue, particularly in fresh biopsy material and tissue blocks stored for less than 5 years. While the diagnostic sensitivity of material containing only spems was higher using IHC, PCR detection was possible in IHC negative liver tissue and in patients with negative serology. Our results support the view that spems do not contain parasitic DNA or viable cells of the parasite. spems thus most probably do not directly contribute to metastasis formation during AE.     

First detection of Echinococcus multilocularis infection in two species of nonhuman primates raised in a zoo: A fatal case in Cercopithecus diana and a strongly suspected case of spontaneous recovery in Macaca nigra

The causative parasite of alveolar echinococcosis, Echinococcus multilocularis, maintains its life cycle between red foxes (Vulpes vulples, the definitive hosts) and voles (the intermediate hosts) in Hokkaido, Japan. Primates, including humans, and some other mammal species can be infected by the accidental ingestion of eggs in the feces of red foxes. In August 2011, a 6-year-old zoo-raised female Diana monkey (Cercopithecus diana) died from alveolar echinococcosis. E. multilocularis infection was confirmed by histopathological examination and detection of the E. multilocularis DNA by polymerase chain reaction (PCR). A field survey in the zoo showed that fox intrusion was common, and serodiagnosis of various nonhuman primates using western blotting detected a case of a 14-year-old female Celebes crested macaque (Macaca nigra) that was weakly positive for E. multilocularis. Computed tomography revealed only one small calcified lesion (approximately 8 mm) in the macaque's liver, and both western blotting and enzyme-linked immunosorbent assay (ELISA) showed a gradual decline of antibody titer. These findings strongly suggest that the animal had recovered spontaneously. Until this study, spontaneous recovery from E. multilocularis infection in a nonhuman primate had never been reported.