In utero transmission of Encephalitozoon

cuniculi strain type I in rabbits

P. J. R. Baneux1 & F. Pognan2
1
Comparative Medicine, PŽ zer Global Research and Development, Amboise, France and
2
Present address: Molecular and Investigative Toxicology, AstraZeneca Safety Assessment,
Wilmington, DE, USA

Summary
Pregnant rabbits were serologically diagnosed as having been infected with Enc e ph a li to zo o n
c un iculi. At necropsy at 28 days of gestation, does, placentas and fetuses were found to be
infected with E. c unic uli strain type I as evidenced by using the nested-polymerase chain
reaction (PCR) technique, thereby con®rming vertical transplacental transmission.

Keywords Enc e ph a lit o zo o n c unic uli ; rabbits; vertical transm ission; transplacental infection

Microsporidia are obligate intracellular pro- are required for a de®nitive diagnosis
tozoa that infect a large number of inverte- (Franzen & Muller 1999 ).
brate and vertebrat e animals (Mathis 2000 ). We examined the presence of E. c unic uli in
T he microsporidial genus Enc e ph a lito zo o n pregnant New Zealand White rabbits (O ryc -
contains three known species that are to la gus c uni culus ), their placentas and their
opportunistic parasites of HIV-infected near-term fetuses using a two-step poly-
patients (Weber e t a l. 1994 ). One of these merase chain reaction (nested-PCR ) ampli®-
species, E. c unic uli, occurs in a wide range of cat ion technique, and compared resulting
mam mals and is most likely zoonotic ribosom al DNA (rDNA) sequences with those
(Mathis e t a l. 1997, Franzen & Muller 1999, ®rst published by Vossbrinck e t a l. (1993 ).
Snowden e t a l. 1999 ).
Vertical transmission of E. c unic uli is often
Materials and methods
discussed in publications and has been
claimed in a number of species, e.g. mice All anim al procedures were performed in
(Perrin 1943, Innes e t a l. 1962 ), blue foxes accordance with applicable legislation and
(Mohn e t a l. 1982), rabbits (Hunt e t a l. 1972 ) reviewed by our local animal ethics
and guineapigs (Boot e t a l. 1988 ). However, committee.
others have documented the fai lure to con- Two studies were performed: the ®rst one
®rm transplacental infections in mice and consisted of serological and histopathological
rabbits (Owen & Gannon 1980, Wilson 1986, investigati ons in male rabbits to validate the
Liu e t a l. 1988 ). T hese earlier reports all used detection of E. cunic uli rDNA by the nested-
microscopic, histological and serological PCR technique, and the second study was
diagnosti c methods. Since the discovery of performed in pregnant rabbits to con®rm in
other morphologically indistinguishable ute ro infection and vertical transmission of
Enc e ph a lito zo o n species, additional anti- the microsporidia.
genic, biochemical or nucleic acid analyses
Ra b b its
C o rre spond e nc e to : Ph ilippe J. R. Ba ne ux DVM, P® ze r G lo b a l
R & D, Fra nc e, BP 159, F-37401 Am b o ise, Fra nce Five male and 10 pregnant (28 days gestat ion)
E-m a il: Ph ilippe.Ba neux@P®ze r.co m female rabbits seropositive for E. c unic ul i, as
Accepted 4 October 2002 # Laboratory Animals Ltd. Laboratory Animals (2003) 37, 132–138
Vertical transmission of E. cuniculi in rabbits 133

determined by the carbon immunoassay L13295; L29560; L17072; L13332; L07255;

(India ink) test (CIA) (Wall er & Bergquist Z19563 ). T hey were aligned using the PC-
1982) and the immuno¯uorescent antibody GENE software (Oxford Molecular, England)
(IFA) technique performed by two different and resulted in a 1773 base pair (bp) con-
diagnosti c laboratories, were purchased from tinuous sequence. PCR primers were
a commercial breeder where this organism designed using the PCR-Plan program from
was known to be endemic. All rabbit s were PC-GENE and synthesized by Genosys
euthanized at arrival with an intravenous (England). T he ®rst ampli®cation (PCR1) was
injection of pentobarbital . done using 0.4 mM of each primer P1
0 0

(5 CCT GACGT GGAT GCTAT T CT CT GG 3

Tissue sa m pling on the plus strand at bases 17±40 ) and M1
0 0

Brai ns, livers and kidneys were sampled from (5 TCAATCACT CTACT TATCCGCTATCGG 3
the ®ve male rabbits. Brains, lungs and on the minus strand at bases 1736±1710 ),
kidneys were collected from the pregnant 0.2 mM of each dNT P (Pharm acia Biotech,
anim als, and brains, lungs, kidneys and pla- France), 1 unit of Taq polymerase and Taq
centas, from the fetuses. Organ sam pling was buffer (Life Technologies, France), and 500 ng
optim ized to avoid contaminati on from one of total sample DNA, in a ®nal volume of
anim al to another, and from one organ to 50 ml. Negati ve controls were performed
another within the sam e animal. A careful identically, except that 500 ng Hela cell DNA
Caesarean procedure was used to prevent was used. Once the am pli®ed DNA had been
contaminat ion from does to fetuses. Sam ples cloned, positive controls consisted of the
for DNA extract ion were cut into slices of cloned sequence at 100 plasmid copies per
about 100 to 500 mg and ¯ash-frozen dry at tube and 5 ml of a 100 000th dilution of PCR1
¡ 180 C in liquid nitrogen. All tissues for for the PCR2 reaction. Ampli®cation steps
histopathological exam ination were ®xed in consisted of 35 cycles at 94 C for 45 s, 62 C
10% form alin solution (Li llie). Tissues were for 45 s and 72 C for 2 min, followed by one
embedded in paraf®n wax and 4±5 micron step at 72 C for 7 min. A second ampli®ca-
thick sections were cut and stained with tion step (PCR2) was performed with 5 ml
haematoxylin and eosin. of the ®rst PCR in the sam e mixture as
above, except that primers were P2
0 0

(5 T T GCGGGAT GAGCAGTAGCT GCG 3 ,

Tissue pro c e ssing a nd DNA e xtra c tio n
plus strand at bases 114±136 ) and M2
Samples were homogenized in 10 volumes 0

(5 T GCT GCCACAAACACAACCCG 3 ,
0

(w =v) of 1% sodium dodecyl-sulfate (SDS), minus strand at bases 1571±1551 ). T he

10 mM EDTA, 10 mM Tris-HC l pH: 8.0 buf- ampli®cation consisted of ®ve cycles at 94 C
fer, and incubated for at least 8 h at 50 C in for 45 s, 58 C for 45 s and 72 C for 2 min,
the presence of 50 ml of 1 mg=ml proteinase K followed by 35 cycles at 94 C for 45 s, 62 C
per ml of lysat e. DNAs were extract ed twice for 45 s and 72 C for 2 min, terminated by
by an equal volume of phenol =chloroform = one step at 72 C for 6 min. All ampli®cations
isoamyl alcohol (v:v:v: 25:24:1 ) and ethanol were performed on a `TouchDown’ Hybaid
precipitated. Pellets were suspended in thermocycler (Cera-Labo, France).
10 mM Tris-HC l pH: 8.0; 1 mM EDTA
pH: 8.0 buffer (T E) containing 1 mg=ml of PC R pro d uct ana lysis, c lo ning a nd
RNase =DNase-free (Boehringer-Mannheim). se q ue ncing
DNA concentration and purity were deter- All PCR products and restriction enzyme
mined by optical density at 260 =280 nm. digestions were analysed on 1% agarose gel
in Tris-Borate-EDTA buffer containing ethi-
PC R a nd ne ste d -PC R dium brom ide for UV visualizati on, accord-
T he theoretical E. c unic uli sequence used ing to Sambrook e t a l. (1989 ). Restriction
was built from several cloned ribosomal enzym e digestion was used to check the
DNA (rDNA) sequences of E. c unic uli pub- accuracy of the ampli®ed sequence according
lished in GeneBank (Accession numbers to the rebuilt theoretical sequence (Aat II,
Laboratory Animals (2003) 37
134 Baneux & Pognan

Bam H I, Bcl I, Hind III, and Xba I as no cut- assayed for PCR1 and PCR2, as described
ting site enzymes and Alu I, Pst I, Sac II and above.
Sph I as single or double cutting site Alternatively, both supernatants and re-
enzymes). T hen, PCR-ampli®ed DNA was suspended pellets (T E buffer) were treated
cloned into pCR2.1 plasmid, using the TA with 1 M NaPO 4 pH: 6.0 to obtain a ®nal
cloning kit from Invitrogen (T he Nether- concentrat ion of 50 mM, and 0.5 to 5 units of
0
lands), and transfect ed in INVaF bacteria by chitinase (US-Biological) per 60 ml of urine
electroporati on using an EEM -600 (BT X, San and incubated overnight at 37 C, followed by
Diego, USA). Bac teria were plated on ampi- incubation with proteinase K at 55 C for 2 h.
cillin and kanam ycin agar plates, containing Sonicat ion for 2 min with a probe at max-
IPT G and X-Gal for blue±white selection of im um power on both pellets and non-sepa-
positive colonies. Plasm ids displaying a cor- rated urines has also been tried prior to
rect restriction pattern were sequenced using chitinase incubation. T he solutions were
the dideoxynucleotide method. Sequencing phenol =chloroform extracted and ethanol
was performed by autom ated ¯uorescence precipitat ed. Pellets were suspended T E-
methods using the ABI 373 (Applied Biosys- RNase-DNase free as above and assayed for
tems). T he polym erase enzym e used was Taq nested-PCR.
FS (Perkin Elm er).

Atte m pts to d e te c t rDNA in urine

Results
Urine of males 1, 2 and 5 was collected at
necropsy by bladder puncture. Animals 3 and Se ro lo gy a nd h isto pa th olo gy
4 had empty bladders. Urines were either Serological test results are shown in Table 1.
centrifuged at 25 000 rpm in a Beckm an Animal 2, positive on CIA and negative on
SW41 rotor or used directly. In the cen- IFA, was later also determ ined as positive by
trifugation case, both supernatant and pellets using the PCR technique. Animal 5 showed a
were assayed. Pellets were suspended in lysis weak positive result on CIA. Although no
buffer (10 mM Na3-EDTA, 0.5% SDS and special stains were used, histopathological
50 mM Tris-HC l pH: 8), containing 6 mg=ml exam inations revealed lesions consistent
proteinase K (Boehringer Mannheim) and with E. c unic uli infection: mark ed to severe
incubated overnight at 55 C. T his was fol- granulomatous in¯am mation in the brain,
lowed by two phenol =chloroform extract ions mild to moderate granulomatous in¯am ma-
and the aqueous phase was ethanol pre- tion in the bile ducts, minimal to mild
cipitated. T he pellets were suspended in interstitial in¯ammat ion in the kidney, and
150 ml of Tris-EDTA pH: 8 buffer (T E), mild nephropathy. T hese results correlated
containing RNase-DNase free enzyme with those obtained by serology, except in
(GIBCO-BRL). T his was used as substrate the case of anim al 2.

Table 1 Comparison between serology, histology and polymerase chain reaction (PCR) analysis of the Ž ve
male rabbits for Encephalitozoon cuniculi infection

Serology Brain Liver Kidneys

Rabbits CIA IFA titres Histo PCR Histo PCR Histo PCR

No. 1 ‡ ‡ (320) ¡ ‡1 ‡ ‡2 ‡ ‡1
No. 2 ‡ ¡ (0) ¡ ‡2 ¡ ¡ ¡ ‡2
No. 3 ‡ ‡ (320) ‡ ‡1 ‡ ¡ ‡ ‡1
No. 4 ¡ ¡ (10) ¡ ¡ ¡ ¡ ¡ ¡
No. 5 ‡ =¡ ‡ (320) ‡ ‡1 ‡ ¡ ‡ ‡1

CIA: carbon immunoassay; IFA: immuno uorescent antibody test; (¡ ) negative; (‡ ) positive; 1 positive in PCR1; 2 positive in
PCR2. IFA titres of 40 or more are positive

Laboratory Animals (2003) 37

Vertical transmission of E. cuniculi in rabbits 135

Fig 1 Alignment of the GeneBank sequences (upper) with the three Vossbrinck sequences (VOSS1, VOSS2 and
VOSS3). They showed almost perfect homologies with the GeneBank sequence. Arrows show the approximate
locations of the primers and the theoretical sizes of the ampliŽ ed fragments are indicated for each couple of
primers. The graph is not to scale

Se q ue nc e a na lyse s of the DNA bands was about 1750 bp. All

Alignm ent of the ®ve Genebank rDNA livers were negative in PCR1. However, the
sequences of E. c uni cul i with the three pub- nested-PCR showed a positive signal in the
lished sequences by Vossbrinck e t a l. (1993 ), liver of rabbit 1 (Fig 3). Table 1 summarizes
showed an alm ost complete homology, the serology results, histology and PCR
0
except for an additional 25 bp in 3 extremity ampli®cations. Histology con®rmed the PCR
of the third Vossbrinck sequence (Fig 1). T he results, except in the case of the livers where
Voss1 sequence accession number is L13295 PCR fail ed to detect the presence of the
and Voss2 and Voss3 have been grouped microsporidia and for animal 2 where his-
under the number L13332, as they overlapped tology did not score lesions consistent with
by 21 bases. T here was 97% homology this parasitic infection in the brain and kid-
between our cloned sequence and the Gene- neys. T he DNA obtain ed from the brain or
Bank sequence, whereas the overall homol- kidneys of all positive animals showed the
ogy between Vossbrinck sequences and sam e restriction pattern, dem onstrat ing that
GeneBank sequence was 99% . Based on the the same target was ampli®ed. Negative
0 0
presence of three 5 -GT T T-3 sequences in rabbi t 2 in PCR1, was positive in PCR2 for
the internal transcribed spacer region of the the brain and kidneys; whereas rabbi t 4,
sequenced cloned DNA, we con®rmed the negative for PCR1, remained negative in
presence of strain type I (rabbit genotype) of PCR2 (Fig 3).
E. c unic uli (Didier e t a l. 1995 ).
PC R o f th e pre gnant ra b b its a nd fe tuse s
PC R o f th e m a le ra b b its T he 10 females were E. cunic uli positive for
Figure 2 shows a positive PCR1 for animals 1, at least one organ in PCR2 (Table 2). T he
3 and 5 in the kidney and the brai n. T he size most frequently positive organ was the brai n

Fig 2 PCR1 ampliŽ cation of the Ž ve male rabbits Fig 3 PCR2 ampliŽ cation of the Ž ve male rabbits
(numbered 1 to 5). N: negative control; B: brain; L: (numbered 1 to 5). N: negative control; B: brain; L:
liver; K: kidney; M: molecular weight ladder (250 bp liver; K: kidney; M: molecular weight ladder (250 bp
repeat, the more intense band is 1000 bp) repeat, the more intense band is 1000 bp)

Laboratory Animals (2003) 37

136 Baneux & Pognan

Table 2 Compilation of polymerase chain reaction (PCR) positive mothers and fetuses

Fetuses

Mothers IFA titres Tissues 1 2 3 4 5 6 7 8 9

10 > 320 BLK – P K – L – – – n

11 > 320 B – – – – – – – – n
12 > 320 B – – – – – – – – n
13 > 320 K L L P – – – – n n
14 > 320 B – P P – P – n n n
15 > 320 BL n n n n n n n n n
16 > 320 B n n n n n n n n n
17 > 320 BL P – P L B BL n n n
18 > 320 LK – – P – – P – – P
19 320 BK BLK – P BLP BL L BLKP n n

Positive organs: B ˆ brain, L ˆ lung, K ˆ kidney and P ˆ placenta. –: negative in any observed organ, n: no fetus. IFA titres of 40
or more are positive

(8 out of 10 ), whereas kidneys and lungs (4 one or more organs, most frequently in the
out of 10) were less frequently infected. placenta (13 =23 ) (Fig 5 gives an exam ple of
However, all positive organs have been sorted placentas from fem ale 19), followed by lungs
out by nested-PCR, as one set of ampli®ca- (10 =23 ). Brains (5 =23) and kidneys (3 =23 ) were
tion gave negative results only. Animals 18 less frequently infected (Fig 6 gives an
and 19 had both kidneys infected (Fig 4), exam ple of fetuses from female 19 ). Females
whereas animal 10 showed only one positive 11 and 12, infected in the brain, had no con-
kidney (not shown). taminat ed fetuses.
Females 15 and 16 were not bearing All at tem pts fail ed to detect the presence
fetuses, and no special stains were performed of rDNA from spores in the urine of infected
to identify possible resorptions. T he other rabbi ts using the same nested-PC R technique,
fem ales had between six and nine fetuses,
giving a total of 59 fetuses (Table 2). T here
were 23 fetuses with E. cunic uli infections in

Fig 5 PCR2 ampliŽ cation of female 19 placentas. M:

molecular weight ladder (250 bp repeat, the more
Fig 4 PCR2 ampliŽ cation of females 18 and 19. B: intense band is 1000 bp); P1: placenta from fetus 1, P2
brain; L: lung; Kr: right kidney; Kl: left kidney; ‡ : from fetus 2, and so on; ‡ : positive control (see
positive control (see Materials and methods) Materials and methods)

Laboratory Animals (2003) 37

Vertical transmission of E. cuniculi in rabbits 137

Fig 6 PCR2 ampliŽ cation of female 19 fetuses (1–7). M: molecular weight ladder (250 bp repeat, the more
intense band is 1000 bp); B: brain; L: lung; K: kidney; ‡ : positive control (see Materials and methods)

with or without the use of chitinase to scrutiny (Hunt e t a l. 1972, Boot e t a l. 1988 ).
disrupt the exospore. However, neither of these reports documents
the simultaneous presence of an En ce ph a li-
to zo o n in the pregnant anim al, the placentas
Discussion and the unborn fetuses, nor do they con®rm
As documented by Boot e t a l. (1988, 2000 ), the species or determ ine the strain of the
the serological tests used to identify Enc e - organism. Possible vertical transmission has
ph a lit o zo o n infection were adequat e and the been suggested in two more species. Anver
histopathological ®ndings correlated well e t a l. (1972) describe the presence of an
with the serological test results. T he failure Enc e ph a lito zo o n in a squirrel monkey (Sa i-
to detect histopathological lesions in animal m iri siu re us) less than 24 h after birth, based
2 was most likely due to limitat ions inherent on light and electron microscopic ®ndings.
in this diagnostic method (Wall er 1977, Also based on morphology, van Rensburg
Gannon 1978 ). T he weak positive result with e t a l. (1991) describe the presence of an
the CIA technique in rabbi t 5 might be Enc e ph a lito zo o n in a still-born foal. In this
attributabl e to a recent infection. study, we clearly show the simultaneous
T he homology between our cloned presence of the parasite rDNA in the preg-
sequence and the Genebank and Vossbrink nant mothers and in some of the fetuses and
sequences con®rmed the presence of E. placentas, dem onstrat ing in ute ro infection.
c un iculi. T hree different strains of E. c uni- It is noteworthy that not all litterm ates were
c uli have been identi®ed based on the num- positive for the observed organs, indicat ing a
0 0
ber of 5 -GT T T-3 sequences in the internal certain randomization in the infection pro-
transcribed spacer region (Didier e t a l. 1995 ). cess. Whether or not these fetuses were
Genotype I (`rabbit strain’), which contains entirely parasit e-free remains uncertain, as
three of those tetranucleotide sequences, was this question would require the extensive
the strain detected in the present study. probing of all organs. However, two does
Many reports of suspected vertical trans- infected in the brain had no apparently
mission of E. cunic uli have been published, infected offspring. T herefore, we have to
yet all are based on circumstantial evidence consider the possibility that vertical trans-
such as the serological or histological diag- mission of E. c uni culi may not be systematic .
nosis of E. c uni cul i infection in offspring Since the diagnosis of Enc e ph a lito zo o n
reared in the absence of potential con- infection in AIDS patients about a decade
tam ination. Only two publications suf®- ago, our knowledge of microsporidia has
ciently describe the gnotobiotic environment increased, other species of Enc e ph a li to zo o n
in which the Caesarean section-derived have been described, and the presence of
offspring were kept to withstand scienti®c different strains has been con®rmed. Based
Laboratory Animals (2003) 37
138 Baneux & Pognan

upon this diversity and the course of infec- Liu JJ, Greeley EH, Shadduck JA (1988) Murine
tion in the different hosts, we will need to re- encephalitozoonosis: the effect of age and mode of
transmission on occurrence of infection. La b o ra -
evaluate the epidemiological charac teristics
to ry Anim a l Sc ie nce 38, 675±9
of these organisms, including their vertical Mathis A, Michel M, Kuster H, Muller C, Weber R,
transmission, and their zoonotic potential. Deplazes P (1997) Two Enc e pha litozo o n c unic uli
Diagnostic techniques are under develop- strains of human origin are infectious to rabbits.
ment in the human medical ®eld (Fedorko Pa ra sito lo gy 114, 29±35
e t a l. 2001, Xiao e t a l. 2001 ). T heir applic- Mathis A (2000) Microsporidia: emerging advances in
ability to the veterinary world might be of understanding the basic biology of these unique
organisms. Inte rna tio na l Jo urna l fo r Pa ra sito lo gy
substantial assistance in this endeavour.
30, 795±804
Mohn SF, Nordstoga K, Moller OM (1982) Experi-
Ac k no w le d gm e nts We would like to thank Dr J.-L. mental encephalitozoonosis in the blue fox:
Le Net, Dipl. ACVP, for his careful analysis and transplacental transmission of the parasite. Ac ta
interpretati on of the histopathology; FrancËoise Rodde Ve te rina ria Sc a nd inavica 23, 211±20
and FrancË oise Borde for their expert technical assis- Owen DG, Gannon J (1980) Investigation into the
tance and James Morelli for editing the photographs. transplacental transmission of Ence ph a lito zo o n
c unic uli in rabbits. La b o ra to ry Anim a ls 14, 35±8
Perrin T L (1943) Spontaneous and experimental
Enc eph a lito zo o n infection in laboratory animals.
Arc h ives o f Pa th o lo gy 36, 559±67
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