Memórias do Instituto Oswaldo Cruz Fundação Oswaldo Cruz, Fiocruz ISSN: 1678-8060 EISSN: 1678-8060 Vol. 97, Num. 7, 2002, pp. 941-945

Mem Inst Oswaldo Cruz, Rio de 
  Janeiro, Vol. 97(7), October 
  2002, pp. 941-945
Enterocytozoon 
  bieneusi (Microsporidia) in Faecal Samples from Domestic 
  Animals from Galicia, Spain  
B Lores+, 
  C del Aguila*, C Arias 
Laboratorio de Parasitología, 
  Facultad de Ciencias, Universidad de Vigo, Vigo, España *Sección 
  de Parasitología, Facultad de CC Experimentales y Técnicas, Universidad 
  San Pablo-CEU, Madrid, España 

  +Corresponding author. Present address: Instituto 
  de Biotecnología, Universidad de Granada, Campus Fuentenueva s/n, 18071 
  Granada, España. Fax: +34-958-243174. E-mail: blores@ugr.es 
  
Received 21 November 2001 

  Accepted 5 June 2002 
This work was supported 
  in part by grants XUGA 30101B97 and Fundación San Pablo-CEU 06/97. 
Code Number: oc02215
In this survey we examined 87 
  domestic animal stool samples in order to detect the possible presence of microsporidia 
  in animals in close contact with humans in Galicia (NW, Spain). The detection 
  of  Enterocytozoon bieneusi spores was confirmed in faecal samples from 
  two dogs and one goat by polymerase chain reaction. None of the positive samples 
  for microsporidia in the staining method were amplified with species-specific 
  primers for Encephalitozoon intestinalis, E. hellem and  E. cuniculi. 
  Four rabbits faecal samples reacted with anti-E. cuniculi serum. Our 
  results could indicate the importance of domestic animals as zoonotic reservoirs 
  of microsporidial human infections. 
Key words: Enterocytozoon bieneusi 
  - domestic animals - faeces - zoonotic origin - Galicia - Spain 
Microsporidia are protozoan parasites 
  belonging to the phylum Microsporidia within which exist over 1000 species classified 
  into approximately 100 genera. These eukaryotic obligate intracellular protozoans 
  have been described infecting every major animal group, especially insects, 
  fish, and mammals (Wittner 1999). Microsporidia have been increasingly recognized 
  as opportunistic pathogens of immunodeficient patients (Weber et al. 1994), 
  especially in Aids patients but it is also becoming increasingly common in immunocompetent 
  individuals (Gainzarain et al. 1998, Lores et al. 2001). 
Although during the last decade numerous 
  data related to the epidemiology of this infection in humans and animals have 
  been accumulated, implying a zoonotic nature of these parasites, direct evidence 
  of transmission from animals to humans are still lacking (Deplazes et al. 2000). 
  
Encephalitozoon cuniculi is 
  probably the most extensively studied mammalian microsporidian and has been 
  reported to infect a wide range of hosts, including common laboratory rodents 
  as well as human and non-human primates. This is the first microsporidian species 
  infecting humans that has been considered a zoonosis (Deplazes et al. 1996, 
  Didier et al. 1996) . 
The first identification of E. 
  intestinalis in mammals other than humans was reported by Bornay et al. 
  (1998) in the faeces of donkeys, dogs, pigs, cow, and goat suggesting that E. 
  intestinalis might also be of zoonotic origin. 
Enterocytozoon bieneusi is 
  the most frequent microsporidian found in humans, especially in Aids patients. 
  It has been associated mainly with chronic diarrhoea, although it has been diagnosed 
  in patients with other forms of immunosuppression and in immunocompetent travellers 
  with self-limited diarrhoea (Weber & Bryan 1994, Sobottka et al. 1995). 
  In addition, this pathogen has recently been detected in other natural hosts 
  such as pigs (Deplazes et al. 1996, Breitenmoser et al. 1999, Rinder et al. 
  2000), cows, goats, pigs, chickens, cats, turkeys (Bornay et al. 1998), rabbits, 
  dogs (del Aguila et al. 1999), and in simian immunodeficiency virus-inoculated 
  monkeys (Tzipori et al. 1997, Mansfield et al. 1997). Consequently, this microsporidian 
  infection may be more common than previously suspected. 
In this study, we used microscopic, 
  immunologic, and molecular methods to detect microsporidial spores in faecal 
  samples of domestic animals from Galicia (NW, Spain). We have designed this 
  survey in order to expand our knowledge concerning the pathogenic role of microsporidia 
  in animals having close contact with humans. 
MATERIALS AND METHODS  
Animal stool samples - A total 
  of 87 faecal samples from 75 domestic animals (22 rabbits, 17 dogs, 10 cats, 
  4 pigs, 2 donkeys, 10 horses, 7 goats, and 3 cows) were collected during a study 
  conducted in rural villages in the provinces of Pontevedra and Ourense (NW, 
  Spain). Samples were stored in 10% formalin for several months. At the time 
  of this study, samples were concentrated by ethyl acetate, and stored at 4ºC 
  until used. Unpreserved samples were aliquoted and conserved at -80ºC for 
  molecular study. 
Light microscopy - The samples 
  were stained with the Weber's chromotrope to investigate microsporidia as described 
  (Weber et al. 1992). A search for ova and parasites was carried out, including 
  ethyl acetate sedimentation, Ziehl-Neelsen stain and a specific monoclonal antibody 
  test for Cryptosporidium species (Merifluorâ, Meridian Diagnostics, 
  Inc.). 
Microorganisms - E. cuniculi 
  (ECLD), E. hellem (CDC:V257) and E. intestinalis (CDC:V297), 
  were cultured on E6 monolayers to be used as controls (Visvesvara et al. 1995). 
  
Indirect immunofluorescence test 
  (IIF) - Spores of E. cuniculi (ECLD), E. hellem (CDC:V257) 
  and E. intestinalis (CDC:V297) were harvested from culture supernatants 
  and processed as described (Visvesvara et al. 1991). The IIF test was performed 
  with specific rabbit antisera on smears from faecal and urine sediment samples. 
  Also, IIF was performed on control faeces known to contain E. bieneusi. 
  
DNA extraction, purification and 
  PCR amplification - DNA was extracted from unpreserved faecal samples by 
  bead disruption of spores, followed by digestion with proteinase K, as previously 
  described (da Silva et al. 1999). PCR inhibitors were removed using the QIAquick 
  PCR Kit (Qiagen, Chatsworth, CA) according to the manufacturer's instructions. 
  
Microsporidial SSU-rRNA coding regions 
  were amplified using the following species-specific primers: EBIEF1/EBIER1 for 
  E. bieneusi (da Silva et al. 1996), SINTF/SINTR for E. intestinalis 
  (da Silva et al. 1997), ECUNF/ECUNR for E. cuniculi and EHELF/EHELR for 
  E. hellem (Visvesvara et al. 1994). A GenAmp kit (Perkin Elmer Cetus, 
  Norwalk, CT, USA) was used for PCR amplification according to manufacturer's 
  directions. Concentration of each primer was 0.1 µg per 50 µl final 
  PCR reaction volume containing 1 or 0.1 µl of the purified stool sample 
  extract. The positive controls used included 0.003 ng per 50 µl PCR reaction 
  of the corresponding cloned SSU-rRNA coding region. Conditions for PCR reactions 
  were: denaturing at 94ºC for 30 sec in all cases, annealing at 45ºC 
  for 30 sec for E. intestinalis primers and at 55ºC for 30 sec for 
  the rest of primers, and extension at 72ºC for 90 sec. In all cases, 35 
  cycles were completed. Amplification products were analysed after electrophoresis 
  in a 2% agarose gel and visualized by staining with ethidium bromide. 
Test for PCR inhibitors in purified 
  samples -Todetect samples with low DNA yield and to control the inhibitory 
  effect in the PCR reaction, purified samples were spiked with 0.003 ng 
  of the corresponding cloned SSU-rRNA. Amplification of a band of the correct 
  size indicated the removal of PCR inhibitors in the DNA purification process. 
  
RESULTS 
Light microscopy - With 
  Weber's chromotrope-based stain, 10 samples showed a low number of spores that 
  stained pinkish red with the Weber's stain and measured approximately 1-1.5 
  µm in length in some cases and about 2.5 µm in others. Spores observed 
  in two rabbit and in one donkey samples were larger and morphologically distinct 
  from spores found in the rest of animal stools. Clusters of microsporidia-like 
  spores within a vacuole inside epithelial cells were also detected in the faecal 
  smears of one dog and one goat. A low parasite burden determined by light microscopy 
  was detected in all cases, this requiring the examination of several slides 
  per sample. 
In 10 (45.4%) rabbits samples, Eimeria 
  spp. was identified, coinciding with two microsporidia positive-samples. Trichuris 
  sp. ova were detected in two dog samples that had been found positive to microsporidia. 
  Trichostrongylus sp. ova were identified in one microsporidia-positive 
  goat sample. 
Parasites found in the animal faecal 
  samples are summarized in the Table. 
  Enteric parasites were identified in most of animals studied and polyparasitism 
  was a generalized characteristic in all samples. Nematodes predominated over 
  Protozoa, and larvae and/or ova in different developmental stages were frequent 
  in herbivores (donkeys, horses, goats and cows). 
IIF - Microsporidia spores 
  present in the smears from the two microsporidia-positive rabbits reacted with 
  the polyclonal anti-E. cuniculi serum in IIF. The few fluorescent spores 
  found appeared isolated and therefore difficult to diagnose. 
PCR amplification - PCR was 
  performed on unfixed faecal samples from the microsporidia-positive animals. 
  The DNA isolated from the five faecal samples from two dogs and one goat was 
  amplified with E. bieneusi specific primers and showed a diagnostic 
  band of 607 bp in the agarose gels (Figure). 
  No amplification was found in any of the five samples when species-specific 
  primers for E. intestinalis, E. hellem, and E. cuniculi 
  were used. 
The donkey faecal samples that showed 
  Encephalitozoon-like spores did not react with any of the primers used. 
  To detect the possible PCR inhibitors in donkey faeces, we tested samples using 
  higher dilutions of template (da Silva et al. 1997) although no amplification 
  was observed. 
DISCUSSION 
The sources of microsporidia human 
  infections and modes of transmission, especially for E. bieneusi, remain 
  uncertain. Persons or animals infected with microsporidia release spores into 
  the environment via faecal, urine, and respiratory excretions, which all could 
  be possible sources of infection (Bornay et al. 1998). Although, the microsporidia 
  have been documented in one waterborne outbreak, the role of animals as the 
  cause of contamination was not elucidated. In food, surface contamination is 
  associated with the faecal-oral pathogens, and some data are available to indicate 
  that animal wastes constitute a major source of contamination (Slifko et al. 
  2000). 
Epidemiologic data are limited, and 
  there are scarce data on animals as potential reservoir hosts for mi-crosporidia 
  that infect humans (Deplazes et al. 1996, Black et al. 1997, Mansfield et al. 
  1997, Bornay-Llinares et al. 1998, Didier et al. 1998). 
In Spain, del Aguila et al. (1999) 
  were the first to report the detection of E. bieneusi in faecal 
  samples from wild and domestic rabbits and in domestic dogs, suggesting the 
  possible zoonotic role of E. bieneusi, to date considered exclusively 
  a human parasite. 
E. bieneusi was identified 
  by PCR in pig faecal samples with a prevalence of 35% (Breitenmoser et al. 1999) 
  and, although it seems to be a common parasite in swine, no genotypes that have 
  been identified in humans were found. However, a recent study (Rinder et al. 
  2000) indicate a close relationship between E. bieneusi strains from 
  humans and pigs, suggesting the absence of transmission barrier between pigs 
  and humans for this parasite. 
In the present study,using Weber's 
  chromotrope stain, IFI and PCR techniques, we detected microsporidian spores 
  in 10 faecal samples from domestic animals (2 rabbits, 2 dogs, 1 goat, and 1 
  donkey) from rural villages in Galicia. Microcopic examination of spores inside 
  the epithelial cells of faecal samples from the animals studied (dog and goat) 
  suggests that these animals could be hosts of E. bieneusi. 
However, we do not know whether the 
  presence of E. bieneusi in the faecal samples are the result of an active 
  infection or a simple passing of the spores through the digestive tract (del 
  Aguila et al. 1999). Nevertheless, these results demonstrate that these animals 
  harboured E. bieneusi spores and shed them into the environment. This 
  finding will be useful to establish other possible transmission routes and to 
  broaden the knowledge of the human epidemiology of this infection. 
To date, only E. cuniculi 
  infection is considered a zoonosis (Deplazes et al. 1996, Didier et al. 1996). 
  There are three different strains of E. cuniculi identified by Western 
  blot analysis of spore antigens and by random amplification of polymorphic DNA, 
  as well as by determination of differences in the rDNA intergenic spacer region: 
  strain type I includes rabbit isolates, strain type II includes murine isolates 
  and strain type III, dog isolates (Didier et al. 1995). The canine and rabbit 
  strains have been identified in three and six patients, respectively (Deplazes 
  et al. 1996, Didier et al. 1996, Mathis et al. 1997, Weber et al. 1997). 
The above results show that in two 
  of the rabbits E. cuniculi spores were identified by IFI. It is unknown 
  whether the presence of the E. cuniculi spores released in the faeces 
  of these animals might signify colonization or infection by the parasite, or 
  simply passage through the digestive tract. This could also be a disseminated 
  infection and thus the spores appeared in the faeces by urine contamination. 
  
In the faeces of a donkey, we found 
  spores with a size compatible with the microsporidia of the genus Encephalitozoon, 
  but these had no amplification by PCR of the DNA studied. The application of 
  the PCR technique in the analysis of faecal samples should be performed with 
  caution to avoid false negatives. If the parasite-DNA concentration is low, 
  the amplification requires large volumes of sample in the reaction. Nevertheless, 
  the presence of certain samples of PCR inhibitors allows adequate amplification 
  only when these are more diluted (da Silva et al. 1997, Ommbruck et al. 1997). 
  In this case, despite the repeated reactions with different DNA dilutions, amplification 
  does not occur with the species-specific primers assayed. 
Furthermore, the high degree of parasitism 
  found should be emphasized, these being multiple infections mostly by nematode 
  parasites. Microsporidians are intracellular parasites ranked among the protists, 
  which means that they are eukaryotic and unicellular. They exhibit a number 
  of important, unique features which are the basis for the systematic ranking 
  of microsporidia, which are knowledged to constitute a phylum of their own (Wittner 
  1999). In particular, in the area where this study was made, helminthosis predominates 
  over protozoosis, precisely the opposite of the situation found in other surveys 
  in Spain, and consistent with the research performed on the presence of intestinal 
  parasites in diverse human populations and in other mammals in Galicia (Arias 
  1980). The area studied (NW, Spain), with a sub-humid Mediterranean weather-type 
  with an Atlantic tendency (Carballeira et al. 1983), includes an environment 
  favourable for the development of infective stages of different nematode species. 
  
The presence of microsporidia in 
  the natural infections of animals are important for the study of clinically 
  significant disease in humans (Rabeneck et al. 1995). With the recent improvements 
  in diagnostic methods for detecting microsporidia, reports are being published 
  about this infection, and as more is learned about the epidemiology, immunology, 
  and pathology of microsporidiosis, advances in the prevention and control of 
  microsporidiosis are more likely in susceptible mammalian hosts (Didier et al. 
  2000). 
The detection of new Enterocytozoon 
  genotypes in faecal samples of domestic animals, together with recent reports 
  of detection of E. bieneusi in environmental samples, suggests that microsporidia 
  of the genus Enterocytozoon is ubiquitous and has many genotypes in various 
  infected animal species (Mathis et al. 1999). 
In zoonotic studies, it is important 
  to investigate all the possible reservoirs for each causal pathogenic agent 
  in any geographical area (Lores et al. 1994) and thus it is crucial to confirm 
  the zoonotic role of E. bieneusi, as this fact may change the established 
  idea of E. bieneusi as a human parasite and possibly characterize it 
  as a zoonotic parasite. As microsporidia are released into the environment via 
  stool, urine and respiratory excretion, possible sources of infection may be 
  persons or animals infected with this group of parasites (del Aguila et al. 
  1999). 
Finally, the role of animals in the 
  transmission of microsporidial human infections requires further study. Although 
  animals have been implicated in some opportunistic infections among HIV-infected 
  persons, the overall risk of transmission from contact with domestic animals 
  is unknown, but may be further reduced. Nevertheless immunodeficient patients, 
  especially with Aids, should be informed with practical suggestions designed 
  to reduce this low risk. 
ACKNOWLEDGMENTS 
To Florentina Martinez and Adel Vázquez 
  for help in collecting samples. To Dr A Osuna, Instituto de Biotecnología, 
  Universidad de Granada, for helpful discussion. 
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