Alexander Mathis

Zoonotic Potential of the Microsporidia

SUMMARY Microsporidia are long-known parasitic organisms of almost every animal group, including invertebrates and vertebrates. Microsporidia emerged as important opportunistic pathogens in humans when AIDS became pandemic and, more recently, have also increasingly been detected in otherwise immunocompromised patients, including organ transplant recipients, and in immunocompetent persons with corneal infection or diarrhea. Two species causing rare infections in humans, Encephalitozoon cuniculi and Brachiola vesicularum, had previously been described from animal hosts (vertebrates and insects, respectively). However, several new microsporidial species, including Enterocytozoon bieneusi, the most prevalent human microsporidian causing human immunodeficiency virus-associated diarrhea, have been discovered in humans, raising the question of their natural origin. Vertebrate hosts are now identified for all four major microsporidial species infecting humans (E. bieneusi and the three Encephalitozoon spp.), implying a zoonotic nature of these parasites. Molecular studies have identified phenotypic and/or genetic variability within these species, indicating that they are not uniform, and have allowed the question of their zoonotic potential to be addressed. The focus of this review is the zoonotic potential of the various microsporidia and a brief update on other microsporidia which have no known host or an invertebrate host and which cause rare infections in humans.

High prevalence of Echinococcus multilocularis in urban red foxes ( Vulpes vulpes ) and voles ( Arvicola terrestris ) in the city of Zürich, Switzerland

Over a period of 26 months from January 1996 to February 1998, 388 foxes from the city of Zürich, Switzerland, were examined for intestinal infections with Echinococcus multilocularis and other helminths. The prevalence of E. multilocularis in foxes sampled during winter increased significantly from 47% in the urban to 67% in the adjacent recreational area, whereas prevalence rates of other helminths were similar in both areas. Seasonal differences in the prevalence of E. multilocularis were only found in urban subadult male foxes which were significantly less frequently infected in summer than in winter. The distribution of the Echinococcus biomass, as expressed by worm numbers per fox was overdispersed in 133 infected foxes randomly sampled in winter. Ten of these foxes (8%) were infected with more than 10,000 specimens and carried 72% of the total biomass of E. multilocularis (398,653 worms). Prevalences did not differ significantly in these foxes in regard to age and sex but worm burdens were significantly higher in subadult foxes as compared with adult foxes. In voles (Arvicola terrestris) trapped in a city park of Zürich, E. multilocularis metacestodes were identified by morphological examination and by PCR. The prevalence was 20% among 60 rodents in 1997 and 9% among 75 rodents in 1998. Protoscoleces occurred in 2 of the cases from 1997. The possible risk for human infection is discussed with respect to the established urban E. multilocularis cycle.

Identification of taeniid eggs in the faeces from carnivores based on multiplex PCR using targets in mitochondrial DNA

A multiplex polymerase chain reaction (PCR) was evaluated for the identification of morphologically indistinguishable eggs of the taeniid tapeworms from carnivores using primers targeting mitochondrial genes. The primers for Echinococcus multilocularis (amplicon size 395 bp) were species-specific as assessed by in silico analysis and in the PCR using well-defined control samples. The design of primers that specifically amplify DNA from E. granulosus or Taenia spp. was not possible. The primers designed for E. granulosus also amplified DNA (117 bp) from E. vogeli, and those designed for Taenia spp. amplified products (267 bp) from species of Mesocestoides, Dipylidium and Diphyllobothrium. Nevertheless, as our diagnostic approach includes the concentration of taeniid eggs by sequential sieving and flotation, followed by their morphological detection, this non-specificity has limited practical importance. Sequence analysis of the corresponding amplicon can identify most of the described E. granulosus genotypes. Taenia spp. can be identified by direct sequencing of the 267 bp amplicon, or, for most species, by restriction fragment length polymorphism (RFLP) analysis. The multiplex PCR was readily able to detect 1 egg (estimated to contain 7000 targets, as determined by quantitative PCR). Having been validated using a panel of well-defined samples from carnivores with known infection status, this approach proved to be useful for the identification of taeniid eggs from both individual animals and for epidemiological studies.

An improved test system for PCR-based specific detection of Echinococcus multilocularis eggs

For the sensitive detection of eggs of Echinococcus multilocularis in fox faeces by PCR we have evaluated a method based on the previous concentration of helminth eggs by a combination of sequential sieving of faecal samples and flotation of the eggs in zinc chloride solution. The eggs were microscopically detected in the fractions retained in 40 and 20 microns mesh sieves. DNA of the taeniid eggs retained in the 20 microns sieve was obtained after alkaline lysis and PCR was performed using E. multilocularis species-specific primers. Compared to the parasitological findings after examination of the small intestines of the foxes, the specificity of the PCR was 100% (no false-positive result with 20 foxes free of E. multilocularis) and the sensitivity was 94% (33 positive results from total 35 foxes proven to be infected with E. multilocularis). Both false-negative results were obtained with faeces from foxes harbouring immature worms. Using faecal volumes between 2 and 20 ml, no inhibition of PCR was observed as was demonstrated by the amplification of size-modified target in parallel reactions. The tests were undertaken with fresh faeces stored in 70% ethanol, but egg detection by PCR was also possible after inactivation of eggs by freezing the faeces at -80 degrees C for one week or by incubation at +70 degrees C for 2 h.

Molecular Epidemiology of Encephalitozoon cuniculi and First Detection of Enterocytozoon bieneusi in Faecal Samples of Pigs

E. cuniculi is the only microsporidial species infecting humans with well documented animal reservoir hosts. However, only few data are available concerning the distribution of E. cuniculi and its subtypes [3] which may be of epidemiological importance. We report on the epidemiological situation of E. cuniculi in humans and rabbits in Switzerland and on the fvst detection of E. bieneusi in faecal samples of pigs. MATERIALS AND METHODS. Specimens of animals and humans were screened for microsporidial spores with the chromotrope stain [7]. Specific antibodies against purified E. cuniculi spores from in vitro cultures were detected in IFAT or ELISA (P. Deplazes unpubl.). Encephalitozoon-isolates from animals or humans were cultivated in MRCJ cells. The species were determined by Western blot (WB) analysis and by ribotyping of the SSU rRNA gene [2]. Characterization of E. cuniculi subtypes included WB analysis, random amplified polymorphic DNA (RAPD) and the determination of the rDNA internal transcribed spacer (ITS) sequence as described 161. E. bieneusi was detected in faecal samples of pigs using two different primer pairs [1,5]. Both strands of one of these amplicons [5] were sequenced with an automated DNA sequencer (Pharmacia). RESULTS AND DISCUSSION. In Swiss rabbit populations, specific antibodies against E. cuniculi spores were detected in 7.5% of 292 healthy rabbits from 126 owners and 85% of 72 rabbits with neurological symptoms from 48 owners. From 20 serologically positive rabbits, the parasites were successfully cultivated in MRCJ cells in 19 cases. Furthermore, 86 wild red foxes and 212 dogs were negative and out of 45 cats only one (with ocular symptoms) showed a specific antibody reaction.

Spatial and temporal aspects of urban transmission of Echinococcus multilocularis

High prevalences of Echinococcus multilocularis have been reported from foxes of the city of Zurich, Switzerland. In order to characterize transmission in urban areas, a coproantigen ELISA was evaluated for diagnosing the infection in fox faecal samples collected in the environment. In addition, trapped rodents were investigated for the presence of metacestodes. Faecal samples could reliably be classified as being of fox origin by assessing physical properties as shown by the different parasite spectra of putative fox and dog faecal specimens. From the total of 604 tested putative fox faecal samples 156 (25.8%) were positive in the ELISA with a distinct increase in the proportion of positive samples from the urban to the periurban zone. Furthermore, samples collected in the border zone had significantly more coproantigen-positive results during winter. Prevalence of E. multilocularis in rodents was 9.1% (81/889) for Arvicola terrestris (with 3.5% of the animals harbouring between 14 and 244400 protoscoleces) and 2.4% (2/83) for Clethrionomys glareolus. E. multilocularis-infected A. terrestris were found in 9 of 10 trapping sites in the border zone. The high infection pressure in the periphery of urban areas might pose a risk for infection with E. multilocularis for both domestic carnivores as well as for urban inhabitants. Interventions into the cycle aiming at reducing the infection pressure should therefore focus on these areas.

High prevalence of Enterocytozoon bieneusi in swine with four genotypes that differ from those identified in humans.

The microsporidial species Enterocytozoon bieneusi is found among immunocompromised, particularly HIV-infected, patients with chronic diarrhoea, and rarely also among immunocompetent persons with self-limited diarrhoea. Only recently, E. bieneusi was detected in 4 pigs in Switzerland raising the question of a potential zoonotic nature of this parasite. We examined faecal samples of 109 pigs, 24 cows, horses and red foxes each for the presence of E. bieneusi by PCR and compared these isolates with isolates obtained from stool samples of 13 HIV-infected patients living in Switzerland. In animals, E. bieneusi was only identified in pigs with a prevalence of 35%. Analysis of the rDNA internal transcribed spacer (ITS) sequence allowed the classification of E. bieneusi from 28 pigs into 4 distinct genotypes which grouped very closely (identity 96.3-98.8%) together with 2 of the 3 human-derived E. bieneusi genotypes. Hence, E. bieneusi seems to be a common parasite in swine, but no genotypes were identified that were found in humans. Nevertheless, swine might serve as a new animal model for enterocytozoonosis.

Polymerase chain reaction for detection of patent infections of Echinococcus granulosus (''sheep strain'') in naturally infected dogs

Polymerase chain reaction (PCR) for the identification of eggs of the tapeworm Echinococcus granulosus (“sheep strain”) was evaluated with primers derived from mitochondrial sequences. Specificity of these primers was confirmed by investigating DNA of other strains of E. granulosus and of 14 helminth species which inhabit the intestines of dogs. This PCR assay was used to investigate 131 purged dogs from Kazakhstan. Eighteen dogs harboured Echinococcus worms, ten of them in mixed infections with Taenia spp. Coproantigen detection was positive in 15 and taeniid eggs could be recovered from 13 of these specimens. Eight of the egg-containing samples were positive in the PCR for E. granulosus and four in a Echinococcus multilocularis -specific PCR revealing one mixed infection. Egg-containing faeces from two dogs harbouring both Taenia spp. and Echinococcus spp. were negative in both PCRs. The combination of egg isolation and PCR will also be of value in epidemiological studies when investigating environmental samples.

The invasive mosquito Aedes japonicus in Central Europe

Complaints about a biting pest led to the recognition of invasive Aedes (Finlaya) japonicus japonicus (Theobald) (Diptera: Culicidae) in Central Europe. Larval collections from cemetery vases revealed a colonized area of approximately 1400 km2 in northern Switzerland spreading into bordering Germany, suggesting that the mosquito has been established in this region for several years. Within this range, larvae of Ae. japonicus were recovered from more containers than the most common resident culicid species Culex pipiens. Possible introduction sites (used tyre yards and international airports) revealed few or no larvae, and the mode of introduction remains unclear. Given the vector potential of this species for arboviruses, implementation of surveillance and control measures should be considered.

Dengue and dengue vectors in the WHO European region: past, present, and scenarios for the future

After 55 years of absence, dengue has re-emerged in the WHO European region both as locally transmitted sporadic cases and as an outbreak in Madeira, driven by the introduction of people infected with the virus and the invasion of the vector mosquito species Aedes aegypti and Aedes albopictus. Models predict a further spread of A albopictus, particularly under climate change conditions. Dengue transmission models suggest a low risk in Europe, but these models too rarely include transmission by A albopictus (the main established vector). Further information gaps exist with regard to the Caucasus and central Asian countries of the WHO European region. Many European countries have implemented surveillance and control measures for invasive mosquitoes, but only a few include surveillance for dengue. As long as no dengue-specific prophylaxis or therapeutics are available, integrated vector management is the most sustainable control option. The rapid elimination of newly introduced A aegypti populations should be targeted in the European region, particularly in southern Europe and the Caucasus, where the species was present for decades until the 1950s.