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请各位老师帮我看看这篇翻译 这是我毕业前要交的一篇外文翻译，请各位老师帮我看看并提出您宝贵的建议。非常感谢！ 由于在这个贴子中不能上传PDF格式的文件因此 由于下载是要花费大家的论坛币的，我会在各位下载后把论坛币返还给各位的。 并且对于提出修改建议的给予一定的论坛币做为补赏您花费的时间。 以下是英文原文： First report of Enterocytozoon bieneusi infection on a pig farm in the Czech epublic
Abstract
Enterocytozoon bieneusi infects humans and animals and can cause life-threatening diarrhea in immunocompromised people.
The routes of transmission and its zoonotic potential are not fully understood. Pigs have been frequently reported to have E.
bieneusi; therefore, we surveyed farm-raised pigs in the Czech Republic to determine its presence and genetic diversity. Spores were
detected by microscopy in the faeces of 65 out of 79 examined animals (82%). A species-specific polymerase chain reaction (PCR)
identified E. bieneusi in 94% of samples. Genotyping based on the ITS regions of the SSU rRNA gene identified that most pigs were
infected with the species-specific genotype F, while two animals had the zoonotic genotype D and two had genotype Peru 9. This is
the first report of E. bieneusi in swine in the Czech Republic, and demonstrated that most infections were with pig-specific
genotypes. Nonetheless, swine may still play a role in the transmission of E. bieneusi to humans.
# 2008 Elsevier B.V. All rights reserved.
Keywords: Enterocytozoon bieneusi; Pigs; Microsporidia; Genotypes; Zoonosis
1. Introduction
Of the 14 species of microsporidia currently known
to infect humans, Enterocytozoon bieneusi (Desportes
et al., 1985) is the most common and important cause of
human microsporidiosis associated with diarrhea and
systemic disease (Didier, 2005). The only published
case of microsporidiosis caused by E. bieneusi in AIDS
patient in the Czech Republic was recorded in 1994
(Ditrich et al., 1994).
Microsporidia spores could be released into the
environment via stool, urine, or respiratory secretions.
They are the infectious stage and can survive in different
environmental matrices such as water, soil, and food
products (Sinski, 2003). The spores of E. bieneusi have
been identified in surface waters in the USA and France
(Dowd et al., 1998; Cotte et al., 1999; Fournier et al.,
2000), and the possible sources of contamination were
humans or animals. Recently, E. bieneusi was also
detected in several wild, domestic and livestock animals
including swine, cattle, goats, birds, rabbits, dogs, cats
and macaques (Deplazes et al., 1996; Kondova et al.,
1998; Breitenmoser et al., 1999; del Aguila et al., 1999;
Mathis et al., 1999; Rinder et al., 2000; Dengjel et al.,
2001; Buckholt et al., 2002; Lores et al., 2002; Reetz
et al., 2002; Fayer et al., 2003; Santı´n et al., 2004, Ha
et al., 2005, Lobo et al., 2006). Sequence analysis of the
ITS region of the rRNA gene showed a close relationship
between E. bieneusi isolates from humans and pigs,
suggestive of an absence of transmission barriers between
these microsporidia isolates (Rinder et al., 2000).
The fact that E. bieneusi was identified in different
animals and water sources have raised public health
concerns about its potential as a zoonotic and waterborne
pathogen (Didier et al., 2004; Cama et al., 2007).
Although vertebrate hosts have been identified for this
microsporidium species infecting humans, the reservoirs
and the modes of transmission of E. bieneusi are
still unknown. Additionally, there is insufficient data to
understand the dynamics of microsporidia infections in
captive and farm animals in Central Europe, including
the Czech Republic.
We report the first survey on the occurrence and
prevalence of E. bieneusi infection on a pig farm in the
Czech Republic and detect its genotypes as a potential
source of human infection.
2. Materials and methods
2.1. Collection of stool samples
We conducted a coprological survey for microsporidia
in a randomly selected farm in the region of
Vysocˇina, Czech Republic during the autumn of 2006.
The selected farm had three different units: two
breeding complexes and a growing complex. Each
breeding complex was divided into two sections: one
with individual pens for furrowing sows and their litters
where piglets (pre-weaners) stay until weaned at 4
weeks of age. The second section was adjacent and
composed of large communal pens for weaned piglets
(starters), where they were kept until reaching 8 weeks
of age. Thereafter, piglets were transferred to the
growing complex (pre-growers), where they were kept
until about 12 weeks of age.
Faeces were collected from animals of different age
categories: pre-weaners, starters, pre-growers and sows.
We randomly sampled 10% of sows, 10% of pregrowers
and three piglets (pre-weaners or starters) per
litter of each sow in this study. The samples were
collected from the floor immediately after defecation
into individually labeled sterile tubes and stored at 4 8C
until processed in the laboratory.
2.2. Detection of microsporidium spores in faeces
Microsporidia were microscopically detected in
faeces using calcofluor white staining (Va´vra et al.,
1993). Briefly, thin smears were made from individual
stool samples and fixed in absolute methanol
and spores were visualized with 0.1% calcofluor
M2R stain (Sigma–Aldrich, St. Louis, MO, USA)
in phosphate-buffered saline (PBS). Evans blue
solution at 0.5% was used to facilitate differentiation.
The slides were examined using UV-light
with a filter wavelength of 490 nm and 1000 magnification.
2.3. Molecular characterization of microsporidia
species and genotypes
2.3.1. DNA extraction
Two to three hundred milligrams of faecal samples
from each sample were homogenized by bead disruption
using a Mini-BeadBeater (Biospec Products,
Bartlesville, OK, USA) for 120 s at a speed
5000 rpm. Total DNA was extracted using the
QIAamp1 DNA Stool Mini Kit (QIAGEN, Hilden,
Germany) following the manufacturer’s instructions
and was kept frozen at 20 8C until PCR amplification.
2.3.2. Molecular identification and
characterization of E. bieneusi
A nested PCR protocol that differentiates E.
bieneusi from other microsporidia commonly found
in humanswas used to amplify a 508 bp fragment of the
small subunit rRNAgene comprised of 122 bp of the 30-
end of the SSU rRNA gene, 243 bp of the ITS and
143 bp of the 50-region of the LSU rRNA gene
(Katzwinkel-Wladarsch et al., 1996). Briefly, primers
sets MSP-1 and MSP-2B, and MSP-3 and MSP-4B
were used for the primary and secondary PCR
amplifications, respectively. PCR amplification consisted
of 35 cycles, of 94 8C for 45 s, 52 8C for 45 s, and
72 8C for 60 s preceded by a denaturation step of 94 8C
for 3 min and followed by a final extension at 72 8C for
7 min. The amplicons were electrophoresed in 2%
agarose gels and visualized with 0.2 mg/ml ethidium
bromide.
All amplified products were sequenced in both
directions using BigDye Terminator chemistries
(Applied Biosystems, Foster City, CA, USA) using
the secondary primers MSP-3 and MSP-4B and
sequenced on the ABI PRISM 3031 (Applied Biosystems)
genetic analyzer. The resulting sequences were
assembled and aligned using the programs ChromasPro
Version 1.32 (Technelysium Pty. Ltd., Qld, Australia)
and Clustal X (ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX/)
and compared with reference sequences from
GenBank.
B. Sak et al. / Veterinary Parasitology 153 (2008) 220–224 221
2.4. Statistics
For statistical evaluation of results, Statistica1,
Release 5.1 Software (Statsoft, Tulsa, OK, USA, 1997)
was used. The Chi-square statistic test for evaluation of
significant differences were utilized.
3. Results
A total of 79 faecal samples were collected from 31
pre-weaners, 16 starters, 17 pre-growers and 15 sows.
At the time of collection, most animals appeared to be in
good health condition, and in two cases (pre-weaner and
pre-grower) diarrhea was inferred from the liquid
consistency of the stools. No association between the
occurrence of diarrhea and E. bieneusi infections in pigs
was found (x2 = 0.1386; df, 1; P > 0.05).
Microsporidia infection was detected in samples
from the three sections of the farm and in all age
categories (Table 1). Stool samples from 65 (82%) pigs
were microscopically positive (range 80–88%).
The PCR analysis of all stool samples detected E.
bieneusi in 74 (94%) of the animals, with very high
percentages among all age categories: all 31 pre-weaners,
15out of16starters, 16out of17pre-growersand12out of
15 sows (Table 1). Sequence analyses of the PCRamplified
product showed that themajority of E. bieneusi
detected in samples were 100% homological with
genotype F (GenBank accession number AF135833),
while four samples belonged to different genotypes: two
were 100% homological with genotype D (samples 50
and 52) and twowith genotype Peru 9 (samples 16 and 80)
(GenBank accessionnumbersAF101200 andAY371284,
respectively),whichwere previously reported in humans.
4. Discussion
In this work the specific diagnosis of E. bieneusi on a
closed pig farm in the Czech Republic was described.
We chose swine because they have been previously
reported to harbour human pathogenic genotypes of E.
bieneusi.
Surprisingly, we identified that 94% of the tested
pigs had E. bieneusi, and that this proportion was
similar among all age categories, and significantly
higher than any previously reported. In 1999, Breitenmoser
reported E. bieneusi in 35% of 109 pigs, and
with much higher occurrence among weaned piglets
(Breitenmoser et al., 1999). Similar to our results, most
of those isolates belonged to genotype F. In an 18-month
survey at a slaughter house in Massachusetts, 32% of
202 finished pigs had E. bieneusi: 18% of them had
microsporidia in their stool samples and the rest in
samples of bile (Buckholt et al., 2002). A small study of
six pigs suffering from severe diarrhea and stunting
identified that four of the six animals had microsporidiosis
(67%) (Rinder et al., 2000).
Although all studies do indicate that microsporidia,
and specifically E. bieneusi can infect swine, it is not yet
clear why there is a broad difference in prevalence
among the different studies. It can be suggested that the
observed differences were the result of different
farming practices from different parts of the world,
although all reported surveys including this one, used
samples from pigs that were raised under intense swine
production practices. These practices include having a
swine exclusive farm, age segregation and age-specific
feeding and enclosed facilities. Besides husbandry
practices, other factors that may affect the presence of
microsporidia in farmed animals can be the health
condition of the herd, age of the animals studied,
location and weather conditions at the farm.
Our results also suggest that it is very likely that
piglets may acquire the infection from their mothers at a
very early age, although the specific routes are unknown.
The faecal oral route has been proposed for acquiring E.
bieneusi infections, and transmission could have
occurred through direct contact with stools from the
sow, nursing from contaminated teats, or ingestion of
faecal material. Nevertheless, additional studies are
needed to ascertain the mechanics of this transmission.
Our study is the first to conduct a methodical survey
that discriminates pigs by age categories within
intensive farming conditions, while also collecting
samples from the sow and its litter. In this case, the close
proximity of the sow with her litter might have resulted
in an overestimation of E. bieneusi positive piglets,
which could have occurred as the result of contamination
with spores from the sow rather than actual
infection of the piglet. Nonetheless, the high percentage
of microsporidia positive animals persisted in piglets
that were weaned and physically separated from their
mothers, reinforcing our findings among pre-weaners.
Our findings also confirm that E. bieneusi genotype F
is the most frequently detected microsporidia in pigs
strengthening the concept of host specificity for this
genotype. Nonetheless, the detection of some genotypes
previously identified in humans suggests that pigs may
play a role, although minor, in the zoonotic transmission
of E. bieneusi.
Although pigs become infected at an early age and
excrete spores lifelong, microsporidia may remain
unrecognized because it is only rarely associated with
severe gastrointestinal symptoms and its detection by
microscopy requires specialized stains that are not part
of the routine coproparasitological diagnosis. Further
epidemiological studies are needed to fully ascertain its
distribution in different geographical settings, weather
conditions and husbandry practices, its economic
impact for the swine industry, and the zoonotic potential
of microsporidiosis in pigs in the Czech Republic.
Acknowledgements
We would like to thank the farm management and
employees to enable us to obtain samples.
This work was supported by the research project of
the Ministry of Education, Youth and Sports of the
Czech Republic (MSM 6007665806), by the grant of
the Grant Agency of the Czech Republic (project no.
523/07/P117) and research project of the Institute of
Parasitology, Biology Centre of the Academy of
Sciences of the Czech Republic (Z60220518).
Table 1
Frequency of E. bieneusi in different age categories of pigs on farms using light microscopy and genotyping
Age category Examination (positive/examined) Genotype F/D/Peru 9
Microscopy PCR
Breeding complex 1 Pre-weaners 19/21 21/21 21/0/0
Starters 10/13 13/13 13/0/0
Sows 7/11 9/11 9/0/0
Breeding complex 2 Pre-weaners 9/10 10/10 8/0/2
Starters 2/3 2/3 2/0/0
Sows 3/4 3/4 3/0/0
Growing complex Pre-growers 15/17 16/17 14/2/0
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