ISRAEL JOURNAL OF
VETERINARY MEDICINE
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VOLUME
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54 (3) 1999
A SURVEY OF ENCEPHALITOZOON CUMCULI INFECHON IN
RABBIT COLONIES
IN ELAZIG, TURKEY: PATHOMORPHOLOGIC AND
SEROLOGIC
(CARBONIMMUNOASSAY TEST) STUDIES
Y. Eroksuz, H. Eroksuz, H. Ozer, A. ַCevik, O.Unver
Firat University, Faculty of Veterinary Medicine, Department of Pathology, 23119, Elazig, Turkey
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Introduction
Materials and Methods
Results
Discussion
Summary
One hundred and fifty rabbits from 15 different colonies were examined for
encephalito- zoonosis both serologically (carbonimmunoassay test) and
pathomorphologically. In total, seropositivity was 65.33%. Gross kidney lesions
consisting of subcapsular depressions were observed in 22 animals all of which were
also seropositive, whereas in seronegative ones no such lesions were detected.
Microscopically, subacute or chronic nonsuppurative and (or) granulomatous
interstitial nephritis of varying severity and distribution was detected in 38 (21.33%)
and focal nonpurulent granulomatous encephalitis in 32 (25.33%) seropositive rabbits
Both lesions were seen in 26 (17.33%) rabbits. Lesions of increasing severity were
associated with detection of pseudocyts in these organs. While 45 seronegative
animals were free from brain and kidney lesions or micro-organisms, mild focal
interstitial nephritis was detected in the remaining seven.
Introduction
Encephalitozoon cuniculi (E. cuniculi) is a microsporidian protozoa that infects that
a wide variety of laboratory and domestic animals. It causes specific tissue reactions
having granulomatous- nonpurulent characteristics in various organs or tissues,
especially the brain and kidney (1, 2). Lagomorph encephalitozoonosis, which is most
common, is generally characterized by mild and subclinical infections, whose
detection depends on diagnostic procedures which include serological, histological or
parasitological examinations. Due to the extended time (4-6 weeks) between the
serologic response and occurrence of the lesions, histopathologic examinations might
give false negative results (3). To date various serodiagnostic methods, including
indirect immunofluorescence (4, 5), skin test (6), complement fixation (7), enzyme
immunoassay (8), dot enzyme-linked immunosorbent assay (9) and carbon
immunoassay-CIA (10), have been used to detect encephalitozoonosis in rabbits. Of
these tests, CIA, whose principle depends on the attachment of carbon particles to the
Fc part of rabbit immunoglobulin G (11), has proven to be both sensitive and reliable
(10, 11). Although several post-mortem histopathologic studies of
encephalitozoonosis have been reported in Turkey (12, 13), no detailed study has been
performed on the its prevalence in the rabbit population. The present study was
designed to document the prevalence of encephalitozoonosis in rabbit colonies in
Elazig province, Turkey, using serological and pathomorphological examinations, and
to describe the distribution of the lesions.
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Introduction
Materials and Methods
Results
Discussion
Materials and Methods
Animals: A total of 150 rabbits (New Zealand white, colored or mrxed breeds, of
either sex) were randomly selected from 15 different rabbit colonies from 1996 to
1997 (Table- 1). Their ages ranged from 14 to 48 weeks and they were clinically
healthy. They were acquired from amateur breeders and departmental facilities of the
Faculty of Veterinary Medicine of Firat University. The animals were maintained is a
closed barrier system (3 or 4 animals per cage) and conventionally in amateur and
departmental facilities.
Histopathology: All the animals, sacrified by cervical dislocation, were necropsied.
Blood samples were allowed to clot and the serum was separated. Representative
tissue samples from major organs including kidney, brain, heart, lung liver, adrenal
glands and small and large intestines were fixed in 10% neutral buffered formalin and
were processed routinely. Tissue sections about 5p,m thick were stained with
hematoxylin and eosin (H&E), van Gieson (vG), Gomori’s or Grocot’s methanamine
silver nitrate (GMS), and Brown and Brenn (B&B) staining methods were also used
on selected sections (14). The presenoe or absence of specific lesions was judged by
examining at least two separate sections of each kidney, comprising cortex and
medulla, and the cerebral hemisphere (two sagittal sections in 2 mm thick from either
sides of the midsagittal plane), and cerebellum. Each tissue size was about 2-2.5 cm2.
Histopathologic diagnosis, which was considered in accordance with a protocol used
by Pakes et al. (6), was judged positive when the protozoa was detected and (or) at
least one granuloma were seen or perivascular cuffing and gliosis were observed in
the brain.
Sera and Serologic test: sera were kept at -20 C until tested. The test kit was a
generous gift from Dr. T. Waller, Testman Laboratory, Uppsala, Sweden. The
procedure was performed as described previously (10). Briefly, serum was diluted in
phosphate buffer saline (PBS) in the ratio of 1/20. Diluted serum was mixed with an
equal amount of antigen suspension (10 p.l) and incubated for 5 minutes at room
temperature. The serum-antigen suspension (10 pl) was mixed with carbon suspension
(10 pl) on a microscope slide. After applying a coverglass, the slides were examined
in a light microscope under oil immersion. In positive results, E. cuniculi spores were
stained black or gray.
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Introduction
Materials and Methods
Results
Discussion
Results
Serologic Studies: The results are presented in Table 1. Briefly, the percentage of
seropositive animals varied greatly among colonies and ranged from 40% to 90%.
The overall seropositivity was 65.33%.
* 1-6: from amateur bredeers
7-15: from departmental facilities
Macroscopic findings: The kidney was the only organ found to have obvious
changes. Out of 150 rabbits, 22 had focal or multifocal depressions on the external
surface of the kidney (Figure 1). These changes were bilateral, grayish-white, and
mild to moderate but they tended to be more apparent after formalin-fixation. On
removal of the capsule, there was no parenchymal tearing. All these rabbits were
seropositive.
Fig. 1: Multifocal and subcortical depression on the external surface of the kidney.
Extrarenal and unrelated lesions included subacute or chronic coccidiosis
associated-cholangiohepatitis (19 cases), striations in the aorta (1 case), focal necrotic
appendicitis and cecocolitis (2 cases) and patchy consolidations in the lung (52 cases).
Microscopic findings: Microscopic changes in seropositive animals as shown in
Table 2 were most frequent in the kidney (38 cases, 25.33%), brain (32-cases,
21.33%), meninges (9 cases, 6%) and the heart (4 cases, 2.67%). Kidney lesions were
characterized by mild to moderate chronic nonpurulent interstitial nephritis.
Interstitial lymphoid infiltrates were either local or diffuse and contained discrete
necrotic tubular epithelium. They were seen both in the cortex and the medulla,
although they were most common in the papi11a renalis. Varying degrees of
interstitial fibrosis with generally scanty mature collagen accompanied by variable
dilation of the collecting tubules and loop of Henle were seen in the cortex. In 8
animals, structures, compatible with E. cuniculi pseudocysts and (or) sporozoites,
were seen in renal tubular cells or tubular lumens without tissue reaction; however,
the lesions inclined to be diffuse in these kidneys. In 7 seronegative animals; mild
interstitial nephritis was recognized by lymphoid cell infiltrations and scanty
collagenous tissue.
Focal or multifocal granulomatous encephalitis was observed in 32 rabbits. Mild to
moderate perivascular mononuclear cell cuffs and (or) glial nodules were seen,
particularly in the vicinity of granulomas (Figure 2).
Fig. 2: Granulomatous meningo-encephalitis characterized by perivascular cuffs, granuloma formation
and lymphocytic infiltration in the choroid plexus. Bar: 150 µm
Small discrete microgranulomas consisting of macrophages, lymphocytes and glial
cells were scattered randomly though most often in the cerebral cortex, and most of
them had no forms visible in the center. These lesions were inclined to be more
diffuse and severe in rabbits whose brain contained single or multiple pseudocysts.
Moderate to extensive meningitis distinguished by infiltration of lymphocytes, plasma
cells and macrophages was also observed in the rabbits with pseudocysts in the
absence of any tissue reaction. In a few rabbits, ependymitis was seen. Four
seropositive animals with brain and (or) kidney lesions also showed separation of the
heart muscle fibers by focal or diffuse mononuclear cell infiltrationt.
Although mononuclear inflammatory foci consisting of granuloma-like aggregates
were seen within the hepatic parenchyma near the portal track and even granulomas
with giant cells in 12 seropositive rabbits, these lesions have not been regarded as
encephalitozoon-associated as all these animals also had lesions of active hepatic
coccidiosis. Similarly, periportal inflammatory reactions were seen in 69 seropositive
and 35 seronegative animals. Except in two cases, all the rabbits with hepatic
coccidiosis were diagnosed serologically as encephalitozoonosis.
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Introduction
Materials and Methods
Results
Discussion
Discussion
There have been many reports documenting the high prevalance of
encephalitozoonosis in rabbits throughout the world using the CIA test (26%, 32%,
35%, 75%), skin test (45%), indirect fluorescent antibody test (28%, 54%),
complement fixation (24%), enzyme immunoassay (50%), and dot enzyme-linked
immunosorbent assay (45%) (4-9, 11, 12, 15- 17). In the present study, a similarly
high seroprevalance (65.33%) concords with these reports.
It is interesting to speculate on how the prevalence of infection can be consistently
high in all studies and yet seronegative animals can persist despite exposure to such a
high and continuous challenge. It is well established (19) that both domestic and wild
adult rabbits can be infected by oral delivery of as little as 50 tissue culture infective
E. cuniculi and that transmission can also readily be effected by intratracheal
administration (3). Earlier studies on transmission of infection in a breeding colony
(5), had shown that young rabbits usually became seropositive in their first few weeks
of life and it was therefore postulated that transmission of infection either occurred
almost immediately post partum or in utero. Several other observations in this paper
merit consideration. Two does seroconverted during the study showing that conditions
did exist for adult-adult transmission. Prior to seroconversion, as with all seronegative
does, all offspring of these two does were seronegative. However after
seroconversion, 70% (19 of 27) of their offspring were seropositive, a percentage
which was higher than that for the offspring of does which were seropositive
throughout the study (27% or 40 of 150). The above observations, should be assessed
along with the observations of Wilson (20) who showed in studies in experimentallyinfected rabbits that uninfected litters could be obtained by cesarian operation
implying that vertical transmission did not occur. Thus it would seem that new- born
rabbits are highly susceptible to infection although the route of infection cannot be
agcertain (i.e., whether oral, respiratory or both). Furthermore, newly-infected adults
excrete greater number of organisms (3), hence it would appear that likelihood of
infection is largely if not solely determined by exposure dose and that there are no
genetic factors predisposing to infection. In most breeding colonies, the majority of
infected does would be expected to be chronically infected and therefore excrete E.
cuniculi only sporadically. Under these circumstances, less than half of their offspring
become infected thus maintaining the prevalence of infection as observed. In wild
rabbits, the infection rate is not so high (21, 22). Even in rabbits which had been most
probably infected in captivity, and released into the wild, the infection does not
persist. The explanation put forward for this is that in the wild, animals are not
reinfected with the parasite they excrete (21). This assumption relies on the
hypothesis that in closed rabbit colonies recycling of parasite is responsible for
persistence. The persistence could also be the result of the mild virulence of the
parasite. Since infection does not exert selective pressure on the host population, it is
also likely that the parasite and host reach a balance in rabbit colonies.
Gross kidney lesions in varying severity were detected only in seropositive but not
seronegative animals, hence, this seemed to be specific for the infection as reported
by many authors (1, 13, 18). With the exception of the mild focal interstitial nephritis
which was detected in 7 seronegative cases, microscopic lesions and detection of
organisms were well correlated with the serologic data. This is consistent with the
finding of 2 cases of interstitial nephritis in seronegative animals (4). This result
indicates that pathomorphologic diagnosis depending only on mild interstitial
nephritis might be misleading. Consequently, the diagnostic criteria proposed by
Pakes (6, 11) were useful in avoiding false positive results. Due to the scattered
distribution of lesions and delay between serologic response and appearance of the
lesions, pathomorphologic diagnosis is prone to false negative results. To minimize
the risk of a false negative conclusion, as many tissue sections as possible have been
examined. However the possibility could not be totally ruled out. High seropositivity
and frequency of lesions have reflected once again that rabbits should not be used as
experimental animals where histopathologic examinations are an endpoint unless
appropriate precautions have been taken either to establish a E. cuniculi-free colony
or at least undertake serological screening to select seronegative individuals for such
studies. Increase in the severity and the distribution of the lesions was associated with
the presence of pseudocysts in these organs, however no inflammatory reaction was
detected around the pseudocysts. This observation would be expected if no foreign
antigens were exposed to the host immune system i.e., all E. cuniculi antigens were
retained within the pseudocyst.
In conclusion, the results of this study indicate that rabbit colonies utilized in
biomedical experiments should be surveyed routinely for the presence of
encephalitozoon infection to ensure the production of reliable data for biomedical
experiments. Serologic results were correlated well with macroscopic lesions in
kidney and microscopic brain lesions. Although histopathologic examinations alone
may give false negative or even false positive results, the CIA was shown to be a
reliable and simple test.
Acknowledgments
The authors sincerely thank Dr. J. Cox, CSL Limited, Victoria, Australia, for critical
reading and Dr. T. Waller, Testman Laboratories, Uppsala, Sweden for kindly
supplying carbonimmunoassay test kit.
References
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Introduction
Materials and Methods
Results
Discussion