Immunohistochemistry Assay to Detect Turkey Coronavirus (TCoV) from Experimentally Infected Poults

Abstract

The objective of this study was to develop a direct immunohistochemical assay to detect TCoV antigens in formalin-fixed paraffin-embedded sections prepared from experimentally infectedpoults. The sections of ileo, ileo-cecal junction and ceca regions from intestine were prepared and submitted to two different primary antibodies, first the non-biotin labeled polyclonal antibody for theindirect method, and second the biotin-labeled polyclonal antibody, both raised against IBV by immunized specific pathogen free chickens. All sections were submitted to immufluorescent assay (IFA), a conventional method, and the results compared. The direct immunohistochemical technique showed a higher frequency of antigen in tissues, especially from the ileo-cecal junction with no difference between results obtained by the conventional method. Finally, the immunofluorescence and all modalities of molecular approaches have been played an important role to the diagnosis and prevention of TCoV infections, although to be precise on infectious disease diagnosis, it is necessary complementary techniques. Here, was standardized the biotin labeled polyclonal antibody as reliable tool to be used as an alternative detection of Turkey Coronavirus.

Full text

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American Journal of Immunology 3 (2): 31-34, 2007
ISSN 1553-619X
© 2007 Science Publications
Corresponding Author: Tereza. C. Cardoso, Departamento de Apoio, Produção e Saude Animal Curso de Medicina
Veterinária Rua Clóvis Pestana, 793. 16.050-680 Araçatuba, SP, Brasil
31

Immunohistochemistry Assay to Detect Turkey Coronavirus (TCoV) from Experimentally
Infected Poults

1Thais Larissa L Castanheira, 2Heitor F. Ferrari, 3Maria Cecília R. Luvizotto and 1 Tereza C. Cardoso
1Laboratório de Virologia, Universidade Estadual Paulista, UNESP Campus de Araçatuba, Faculdade de
Odontologia, Curso de Medicina Veterinária, Araçatuba, SP-Brazil
2Alunos de iniciação científica e Pós-graduação Mestrado em Ciência Animal, Fundação de Amparo à
Pesquisa do Estado de São Paulo, Brazil
3Laboratório de Patologia Animal, Universidade Estadual Paulista, UNESP Campus de Araçatuba,
Faculdade de Odontologia, Curso de Medicina Veterinária, Araçatuba, SP-Brazil

Abstract: The objective of this study was to develop a direct immunohistochemical assay to detect
TCoV antigens in formalin-fixed paraffin-embedded sections prepared from experimentally infected
poults. The sections of ileo, ileo-cecal junction and ceca regions from intestine were prepared and
submitted to two different primary antibodies, first the non-biotin labeled polyclonal antibody for the
indirect method, and second the biotin-labeled polyclonal antibody, both raised against IBV by
immunized specific pathogen free chickens. All sections were submitted to immufluorescent assay
(IFA), a conventional method, and the results compared. The direct immunohistochemical technique
showed a higher frequency of antigen in tissues, especially from the ileo-cecal junction with no
difference between results obtained by the conventional method. Finally, the immunofluorescence and
all modalities of molecular approaches have been played an important role to the diagnosis and
prevention of TCoV infections, although to be precise on infectious disease diagnosis, it is necessary
complementary techniques. Here, was standardized the biotin labeled polyclonal antibody as reliable
tool to be used as an alternative detection of Turkey Coronavirus.

Keywords: TCoV, PEMS, Immunohistochemistry, IFA.

INTRODUCTION


producer in the world with 187 millions of carcases
commercialised during 2006, corresponding to 29% of
the international market. In spite of intensive
production, turkey coronaviral enteritis, the most costly
viral disease has been affected young poults in Brazil in
early 2006 and, the respective outbreak has been
classified as poult enteritis and mortality syndrome-
PEMS, according to previous descriptions
Elsewhere, the clinical signs are characterized as
inappetence, wet droppings, ruffled feathers, decreased
weight gain, growth depression, and uneven flock
growth observed normally from affected breeders,
symptoms also observed in Brazil, for the first time [5].
In fact, Coronaviruses are in the family
Coronaviridae, which are enveloped, positive-stranded
RNA virus that infects a wide range of mammalian and
avian species [6]. So far, Turkey Coronavirus (TCoV)
and infectious bronchitis virus (IBV) belong to antigenic
The Brazilian turkey industry ranks second
[1-4,9].
group III and share antigenic similarity
biological characteristic allows using antigen, as well as,
antibodies raises against IBV, to develop and apply
immunological tools for the TCoV diagnosis [8-10].
The detection of coronavirus direct from
intestinal contents has been usually done by direct
electron microscopy (EM), indirect immunfluorescence
antibody (IFA), ELISA approaches, and molecular
techniques, as RT-PCR or multiplex RT-PCR. Actually,
the IFA and the RT-PCRs are the most important
control measures for the PEMS in the turkey industry
described in USA and Great Britain [3,6] and more
recently in Brazil [9].
Although, the IFA test is routinely applied on fresh or
frozen tissues, it is labours and time consuming when a
large number of specimens must be evaluated. In order
to rapidly diagnose, as well as, effectively control
turkey poult enteritis new techniques must be useful and
specific for clinical samples, especially when the
industry is experiencing an outbreak. In this report, we
developed and applied a useful simple direct
[7]. This

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Am. J. Immunol., 3 (2): 31-34, 2007


32
immunohistochemical technique to detect the TCoV
from formalin-fixed tissues, comparing the results with
those obtained by IFA.

MATERIAL AND METHODS

Experimental infection: Turkey embryos aged 23-25
days of incubation were obtained from commercial
breeder and infected by Brazilian[9] isolate strain. After
72h post-infection the intestine was collected and
divided into three portions: ileo, ileo-cecal junction and
ceca.

Histopathology: Samples of ileo, ileo-cecal junction
and ceca were collected and fixed in 10% neutral
buffered formalin. Than, were embedded in paraffin
blocks, sectioned at 2mm
hematoxylin/eosin (HE) routine method, and finally
examined using light microscope [9].

Primary antibody production:The IgG against IBV
was produced by vaccination of 10 inbred C/O line
white Legorns chickens at one day of age by intra-
ocular route with purified M41 serotype as described
before [11] with some modifications. After two weeks,
the chickens received the second injection of
commercial vaccine, 1ml per bird (H120) by intra-
muscular route, and 21 days after they were bled from
the wing vein. The γ-globulin fraction was prepared by
the salting-out procedure adding 35% (v/v) of
ammonium sulfate (Sigma) and followed by IgY
fraction purification using chromatography separation
on Sephadex-G200 (Sigma).

Biotin labelled antibody production: The purified IgG
was conjugated to biotin (Biotin disulfide N-
hydroxysuccinide ester, Sigma) according to previous
studies [19] with some modifications and used as primary
antibody. The first step was to mix 1mg/ml of chicken
IgG fraction with 250µg/ml of biotin dilute in sodium
borate buffer (Ph 8,8) and left at 4°C during 4h. After
the reaction, the mixture was dialyzed against phosphate
saline buffer (PBS) Ph 7,2 48 h consecutively to
eliminate the non-linked molecules and the antibody
work dilution determined by direct ELISA.

Preparations of sections for staining: Unstained
sections were used for the indirect and direct
immunohistochemical examination just after submitted
to deparafinisation, rehydratation and washes in
buffered saline added by 0,1% Tween 80. First step, the
heat-induced epitope retrieval with citrate buffer (Ph
6.1) for 15 min at 700W was the pre-treatment for viral
and stained with
antigen
formaldehyde fixation. Just before staining, slides were
treated 3 times with hydrogen peroxide (Merck) 50%
during 30 min, 2 times with mixture of hydrogen
peroxide and methanol 30% during 30 min and once
hydrogen peroxide 3% during 1 h to inactivated
endogenous peroxidase,
inflammatory reactions. So, the slides were placed in
saline buffered for 10 min 5 times, consecutively, to
remove the residues between each step of the reaction.
The unspecific bindings were blocked using a variety of
blockers to decrease the background activity. First was
tested dried 15% nonfat milk during 90 min, gelatin 5%
during 60 min, serum albumin bovine (Sigma cat # A-
9647) 5% during 120 min and normal rabbit serum 2%
over night. After established the best condition of
blocking the slides were submitted to procedure
staining.

Indirect Immunohistochemistry:
antibody used in the indirect immunohistochemical
analysis was the same described before, without biotin
labeling. First, the antibody was applied to the slides at
1:100, 1:200 and 1:400 dilutions to determine the
optimal work dilution, made in PBS plus 10% nonfat
dried milk, and incubated over night at 4°C under dark.
The next step was to performer 5 washes of 10 min each
using PBS plus 0.5% Tween 80, and applied the specific
anti-IgG conjugated to peroxidase (HRPO-Zymed goat
anti-chicken linked to peroxidase) diluted 1:500 at PBS
plus nonfat dried milk during 1h at 37°C. After
incubation, the slides were washed and the substrate
made fresh in the dark, by mixing equal volumes of
0.02% hydrogen peroxide and 0.6mg DAB (3,3´-
diaminobenzidine tetrahydrochloride, Gibco BRL), was
added to the slides for 30 min at room temperature. The
reaction was stopped by washing with tap water and the
specific brown color was revealed after counterstained
with Meyer´s hematoxilin. An intensive dark red
deposit was considered positive and the negative
controls consisted of sections treated with buffered
saline instead of primary antibody. The intensity of dark
red deposit was arbitrarily rate on a scale of –
(undetectable) to ++++ (the pattern present at its highest
intensity). Omission of the primary antibody was used
as a negative control for the reaction and the positive
control, infected chicken kidney cells (CKC) infected by
IBV were used.

Direct Immunohistochemistry: Viral antigen was
demonstrated by avidin-biotin complex (ABC) direct
immunoperoxidase method as described before [16] and
with some modifications. The optimal primary biotin
reactivation, normally damaged by
commonly found in
The primary

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33
labeled antibody dilution was determined by titration
(1:100, 1:200 and 1:400) made on PBS plus 10% of
nonfat dried milk covered by 200µl of each dilution
over-night at 4°C in a humidified chamber. After 5
washes, the 100µl/slide of streptavidin-peroxidase
(Sigma cat # S-5512) complex was added and incubated
1h at 37°C. In addition, substrate made fresh in the dark,
by mixing equal volumes of 0.02% hydrogen peroxide
and 0.6mg DAB
tetrahydrochloride, Gibco BRL), was added to the slides
for 30 min at room temperature. The reaction was
stopped by washing with tap water and the specific
brown color was revealed after counterstained with
Meyer´s hematoxilin. An intensive dark red deposit was
considered positive and the negative controls consisted
of sections treated with buffered saline instead of biotin
labeled antibody and the results calculated as described
before. For the parameters to be analyzed the same
procedure described before was used. The IFA was
performed according to previous reports [8].

Statistical Evaluation: Statistical comparisons of
positive results among all the methods were performed
with two-sample t-test and P value determined and
origin 7.0 software were used for data analysis.

RESULTS AND DISCUSSION

Microscopical examination of the intestine (ileo, ileo-
cecal junction and ceca) revealed marked degeneration,
destruction of the villous epithelium, hyperactivation of
the intestinal glands intestinal and lumen filled with
desquamated epithelial cells as well as mucous
exudates. In addition, basal lamina was infiltrated with
mononuclear cells, presented submucosa oedema
compatible with viral enteritis (results not shown).
These findings have been described for PEMS, however
they are common to other enteric pathogens
Regarding to the intestines analyzed here, they were
flaccid, thin-walled, and filled with loose contents, and
the disease was acute, with symptoms appearing and
lasting for about 3 weeks
In order to determine the efficiency of biotin-
labeled antibody produced, the direct ELISA was
performed and the IBV antigen reacted specific against
the respective antibody, producing an optical density pf
1.0 (DO), whilst the non-labeled antibody produced any
OD [9-12]. In this way, the immunohistochemical, indirect
and direct methods, produced results that revealed a
precise detection of TCoV antigens. The work dilution
chosen was 1:200, diluted in PBS plus 10% nonfat dried
milk for the both methods. In addition, the best blocking
(3,3´- diaminobenzidine
[6-8].
condition was the use of serum albumin bovine at 5%,
which gave the best neutralization of non-specific
reactions (Fig. 1A and B).


(A)

(B)
Figure 1: A) Ileo-cecal junction presented positive
browm reaction, +++ cross, classified as
intense (narrowhead); B) Ileo region showed
positive brown reaction on the villus; bar
30µm.

The sections prepared from the ileo presented
same positive results for the immunohistochemical
analyze and IFA. Moreover, in previous reports, it has
been described that ileo-cecal junction is the best region
to detect TCoV from naturally and experimentally
infected poults in agreement with those observed here [3,
5, 9-12].
In fact, the immunohistochemical test, using
the peroxidase system offer advantages over the IFA
method in that: 1) it does not require an ultraviolet
microscope, and 2) the tissues were stained for
immunohistochemical and
microscope evaluation, allowing the observer to
correlate the location, numbers, and intensity of stained
cells with a normal microscopic for pathology
examination.
Herein, the immunohistochemical methods
using the polyclonal antibody demonstrated great
advantage over IFA, where monoclonal antibodies have
been routinely used for the same purpose. However,
diagnosis of TCoV infections based on histopathology
description is not reliable, because other infectious and
non-infectious agents can cause similar symptoms and
microscopic lesions [4].
then re-stained for

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Am. J. Immunol., 3 (2): 31-34, 2007

CONCLUSION

Based on this fact, the RT-PCR and all
modalities of molecular approaches have been played an
important role to the diagnosis and prevention of TCoV
infections. Thereafter, immunohistochemistry assay has
demonstrated viral antigen and may be useful for
differential diagnosis of enteric diseases as an
alternative viable tool for TCoV diagnosis.

ACKNOWLEDGEMENTS
FAPESP - Fundação Amparao à Pesquisa do Estado de
São Paulo (Grant number 05/51484-1 and 06/59769-8).
We are indebt to the technical assistance of Adriana
Petrocelli whom supplied the samples.

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