Complete article

120
CERIBASI (A. O.) AND COLLABORATORS
Diagnosing respiratory syncytial virus using
immunofluorescence and
immunohistochemistry methods in caprine
lungs with bronchopneumonia
S. CERIBASI1, M. OZKARACA2, H. OZER1, A. O. CERIBASI1*
Department of Pathology, Faculty of Veterinary Medicine, Fırat University, 23119 Elazig, TURKEY
Veterinary Control and Research Institute, 23119 Elazıg, TURKEY.
1
2
Corresponding author: [email protected].
SUMMARY
RESUME
The aim of this study was to evaluate the diagnostic interest of direct
immunofluorescence and immunohistochemistry for RSV infection in
goats with bronchopneumonia from the Elazig region. For this purpose,
lungs of 889 slaughtered male hair goats were macroscopically analysed
in order to looking for chronic bronchopneumonia lesions. Among the
146 cases of bronchopneumonia found in which mild lesions (according
to the extent of consolidation) were prominent (65.8%), there were 105
interstitial bronchopneumonia, 41 catarrrhal-purulent bronchopneumonia
and 2 verminous bronchopneumonia according to histological criteria.
The RSV antigens were detected in 7 cases with interstitial chronic
bronchopneumonia using direct immunofluorescence on frozen secions
whereas direct immunohistochemistry on paraffine sections failed to detect
the viral particles. These results show that immunofluorescence on frozen
section was undoubitously more adequat than immunohistochemistry for
RSV detection in situ and they suggest a possible role of RSV in the induction
of interstitial bronchopneumonia in goats.
Diagnostic du VRS chez la chèvre atteinte de bronchopneumonie.
Keywords: Respiratory syncytial virus, goat, lung,
chronic bronchopneumonia, pathology, diagnosis,
immunofluorescence, immunohistochemistry.
L’objectif de cette étude a été d’évaluer l’intérêt diagnostique d’une détection
directe du virus respiratoire syncitial (VRS) par immunofluorescence et
par immunohistochimie chez les chèvres atteintes de bronchopneumonie
provenant de la région d’Elazig. Pour cela, les poumons de 889 boucs
abattus ont été examinés macroscopiquement. Parmi les 146 cas de
bronchopneumonie ainsi identifiés présentant majoritairement (65.8%)
des lésions peu intenses (en fonction de l’étendue de la consolidation), 105
présentaient à l’histologie des lésions de bronchopneumonie interstitielle, 41
des lésions de bronchopneumonie catarrhale et purulente et 2 des lésions de
bronchopneumonie vermineuse. Les antigènes du VRS ont été mis en évidence
dans 7 cas de bronchopneumonie interstitielle par immunofluorescence
directe sur coupes congelées alors que les particules virales n’ont été détecté
dans aucun cas par immunohistochimie sur coupes incluses en paraffine.
Ces résultats montrent que l’immunofluorescence sur coupes congelées est
bien plus adéquate que la technique d’immunohistochimie pour détecter in
situ le VRS et suggèrent un possible rôle de ce virus dans l’induction d’une
bronchopneumonie interstitielle chez la chèvre.
Mots-clés : Virus respiratoire syncitial, chèvre, poumon,
bronchopneumonie chronique, anatomopathologie,
diagnostic, immunofluorescence, immunohistochimie.
Introduction
Respiratory Syncytial Virus (RSV) which is from the
Pneumovirus genus of the Paramyxoviridae family, leads to
significant economic losses by causing respiratory system
infections in cattle and sheep [2]. RSVs in ruminants
have been defined as bovine RSV, ovine RSV and caprine
RSV [8]. An antigenic and structural connection has been
reported between all RSV, but an especially close connection
between bovine RSV and caprine RSV [1, 5, 17, 22]. Previous
studies have indicated that all RSV could cause inter-species
infections [4, 7, 14, 25].
The virus could also be clinically isolated from goats
that did not exhibit respiratory system infection [19]. RSV
infection in goats has been evidenced in many areas of the
world throughout serological and pathological studies [15,
24, 27]. Contamination occurs through droplet infection,
and contaminated animal food and water [23]. Most of the
severe epidemics occur in autumn and winter [26]. Clinical
symptoms such as fever, sluggishness, nasal discharge,
constant and deep breathing, respiratory distress, cough,
anorexia are observed in affected animals [6]. Moderate
clinical symptoms have been emphasized to occur in
experimental RSV infections, whereas intense clinical signs
were encountered during mixed infections with bacterial
agents such as Mannheimia haemolytica [3]. Necropsy
findings are similar in all ruminant RSV infections.
Macroscopically, a mucopurulent exudate in the lumen
of the bronchus and bronchioles are observed together
Revue Méd. Vét., 2013, 164, 3, 120-124
RSV DIAGNOSIS IN GOATS WITH BRONCHOPNEUMONIA
with irregular lobular or diffuse mildly collapsed gray- red
foci in varying sizes in the cranioventral lobes of the lung.
Emphysema may be formed in different regions of the lungs.
Histopathologically, bronchitis, bronchiolitis, thickening
in the alveolar septum with mononuclear cell infiltrations,
lymphoid hyperplasia, hyperplasia in the epithelial cells of
the bronchi and bronchioles, acidophilic inclusion bodies in
the epithelium of the bronchi and bronchioles and syncytial
cells have been reported [11, 13, 19, 24].
Direct and indirect laboratory methods are beneficial in
the diagnosis of RSV. Viral antigens may be directly detected
using methods like indirect immunohistochemistry, direct
immunofluorescence and ELISA [26]. Serological methods
such as complement fixation, neutralization, ELISA, indirect
immunofluorescence, and indirect haemagglutination tests
are used for indirect diagnosis [23].
In this study, it was aimed to determine the prevalence of
RSV antigens in goats in the Elazığ province, Turkey using
direct immunofluorescence and immunohistochelmistry
methods in frozen and paraffin sections obtained from
caprine lungs with bronchopenumonia.
Material and Method
SAMPLE COLLECTIONS
In the study, the lungs of 889 male hair goats (> 1 year
old), that had been slaughtered in a slaughterhouse in
Elazığ between January and June 2011 were investigated.
Macroscopic bronchopneumonia lesions in the cranial and
cardiac lobes were observed in 146 lungs (16.42%). The
severity of bronchopneumonia in cranial and cardiac lobes
was scored according to the extent of consolidation. In
cranial and cardiac lobes, lesions in which 10% or less of lung
volume was affected were evaluated as ‘mild’, those with 10%20% consolidation were evaluated as ‘moderate’, and lesions
with more than 20% of lung volume affected were evaluated
as ‘severe’. Some parts of the injured pulmonary tissues were
fixed in 10% buffered formalin solution and others were
stored at -80°C for frozen sections.
After fixation in 10% buffered formalin, paraffin blocks
were prepared throughout routine procedures. Sections 5 µm
in thickness were obtained and stained with HaematoxylinEosin (HE) and examined under light microscopy.
Additionally, sections were stained with Shorr’s staining
solution according to Page-Green method for the detection
viral inclusion bodies [20].
IMMUNOHISTOCHEMISTRY
Immunohistochemical staining was performed with the
avidin-biotin-peroxidase complex procedure [13], using
commercially available immunoperoxidase kits (Ultravision
Detection System, Antipolyvalent, HRP/DAB, Thermo
Sciencific, Cat No:TP-015-HD). For immunohistochemical
Revue Méd. Vét., 2013, 164, 3, 120-124
121
staining, deparaffinised tissue sections were placed in citrate
buffer solution (10 mM citric acid, pH 6.0) and were kept in
a microwave for 20 minutes for antigen retrieval stage. The
sections were incubated in 70% methanol with 3% H2O2 for 10
minutes to inhibit endogenous peroxidase activity, and were
then washed three times in phosphate-buffered saline (PBS,
pH 7.4). The sections were treated with blocking solution for
10 minutes. After draining the blocking serum, the sections
were incubated with primary antibody [monoclonal mouse
anti-RSV (cat no: bio 031, biox jemelle, Belgium)], diluted
to 1:20, 1:50, 1:100 and 1:200 in PBS at 4°C overnight in a
humidified chamber. After washing in PBS 3 times, sections
were incubated with biotinylated anti-goat polyvalent
secondary antibody for 10 minutes. Then the sections were
washed three times in PBS and treated with the peroxidaseconjugated streptavidin. After another PBS washing step, the
sections were incubated with 3,3’- diaminobenzidine (DAB).
After colour change, sections were washed with tap water
and then counterstained with Mayer’s haematoxylin. Lung
section from a healthy goat was used as negative control.
Furthermore, non-immune mouse serum was used instead
of primary serum in lung sections with pneumonia.
DIRECT IMMUNOFLUORESCENCE
Using a frozen microtome, sections of 6 mm in thickness
were obtained. They were fixed with in 90% acetone solution
and washed with PBS (phosphate Buffer Saline, pH 7.4) and the
surfaces of the samples were coated with monoclonal mouse
anti-RSV fluorescein isothiocyanate (FITC) conjugate (biox
jemelle, Belgium, cat no: bio 032), diluting 20 times with PBSEvans Blue. After incubation for 1 hour at room temperature
(25°C) and washing in PBS, sections were examined under
a fluorescent microscope with 9% glycerol solution. The
negative controls prepared for immunohistochemistry were
also used for the immunofluorescence test.
Results
The severity, type and rates of lesions in
bronchopneumonia and their distribution according
to months in which samples have been collected were
summarized in Table I. Although the severity and
distribution varied, all bronchopneumonia lesions were seen
to be macroscopically characterized by patchy or diffuse,
purple-red or gray, focal or irregular lobular atelectatic foci.
Among the 146 cases, 65.75% of them exhibited mild lesions,
24.66% moderate lesions and 9.59% severe lesions according
to the extent of consolidation. Subpleural emphysema was
observed together with thickening in the interlobular areas
in bronchopneumonia with severe consolidation. Interstitial
bronchopneumonia was the prominent type of pneumonia
found and occurred in 71.92% cases of bronchopneumonia
whereas verminous and catarrhal / purulent types remained
minor (1.37% and 26.71%, respectively). The presence of
RSV antigens was investigated using immunofluorescence
and immunohistochemical methods in the caprine lungs
with interstitial bronchopneumonia (n = 105).
122
CERIBASI (A. O.) AND COLLABORATORS
January
February
March
April
May
June
Total
Number of examined lungs
144
131
155
188
134
137
889
Bronchopneumonia severity
Mild
Moderate
Severe
9
5
3
13
11
1
10
4
3
41
11
4
16
4
2
7
1
1
96 (65.8%)
36 (24.7%)
14 (9.6%)
Bronchopneumonia type
Verminous
Catarrhal and purulent
Interstitial
1
6
10
0
4
21
1
8
8
0
11
45
0
7
15
0
3
6
2 (1.4%)
39 (26.7%)
105 (71.9%)
Table I: Severity, type and distribution of bronchopneumonia lesions in goats (889) according to the month of sample collection.
Using the direct immunofluorescence (DIF) test, RSV
antigens were detected in 7 out of 105 (6.67%) sampled
lungs with interstitial bronchopneumonia. Fluorescence
was observed in the bronchus and the alveolar epithelium
(figure 1); there was spilling of the cellular content into the
lumen, and inflammatory cells invaded the interstitial area
(figure 2). In the afore-mentioned cells, the fluorescence
reactions were seen to be limited to the cytoplasm, were of
varying sizes, and granular in appearance. In addition to
positive staining, autofluorescence reactions were present,
particularly in the capillary vessels. Cytoplasmic granular
fluorescence reactions were not observed in negative control
sections. Using immunohistochemistry, RSV antigens could
not be determined in any of the 105 cases with interstitial
bronchopneumonia.
and lymphoid hyperplasia in the bronchus, bronchioles
and areas adjacent to some alveolar septa. The lumens of
some bronchioles and alveoli were found to have been filled
with neutrophil-rich cellular exudate, and a small numbers
of lymphocytes and macrophages were found to have
contributed to the inflammation, in addition to neutrophils
in some areas. Alveoli adjacent to the bronchioles were seen
to have been destroyed and subjected to atelectasis. No viral
inclusion bodies could be encountered using the Page-Green
method.
Discussion
Only DIF positive interstitial bronchopneumonia cases
were examined microscopically in order to evaluate the
RSV-related changes in the lung sections. Varying degrees
of inter-alveolar septal thickening and fibrosis were the most
prominent microscopic changes. The vast majority of the
alveolar septa were seen to have thickened irregularly due
to an increase in connective tissue together with lymphocyte
and macrophage (at a lesser extent) infiltration. Moreover,
the other prominent lesions included peribronchial and
peribronchiolar mononuclear cell infiltrations together with
hyperplasia in the bronchus and the bronchiolar epithelium
Pneumonia that are microscopically characterized with
bronchitis and bronchiolitis, hyperplasia in the bronchus
and bronchiolar epithelium, thickening in the alveolar
septum, hyperplasia in the epithelial cells of the bronchi and
bronchioles, lymphoid hyperplasia, syncytial cell formations
and cytoplasmic inclusion bodies, are reported to be formed
in RSV infections [6, 13, 24]. Although the microscopic
findings in the presented study are similar to those in
previously described RSV pneumonias, no virus-related
syncytial cells or inclusion bodies could be encountered in
the present study. Occurrence of specific viral lesions like
inclusion body and syncytial cell formations in the lungs
in natural and experimental RSV infections of ruminants
would be inconstant according to many factors such as
Figure 1: RSV direct immunofluorescence test in caprine lung with interstitial bronchopneumonia. Immunofluorescent positive inflammatory cells
(arrowheads) in interstitial areas. Bar: 50 mm.
Figure 2: RSV direct immunofluorescence test in caprine lung with interstitial bronchopneumonia. Immunofluorescent desquamative necrotic cells
(arrowheads) in lumen and epithelium of lung alveoli. Bar: 50 mm.
Revue Méd. Vét., 2013, 164, 3, 120-124
RSV DIAGNOSIS IN GOATS WITH BRONCHOPNEUMONIA
the agent virulence, the virus amount, the duration of the
infection period, age and species of the animal [9, 11, 19].
Furthermore, these lesions have been reported to disappear
when secondary bacterial infections were developing [6].
Diagnosis of RSV infections is based on macroscopic
findings, histopathological findings and detection of viral
antigens using immunohistochemical methods [6, 11].
Furthermore, it has been reported that PCR, cell culturing and
serological methods could also be beneficial [10, 12, 16, 21].
Previous investigations aiming to determine the incidence
of RSV infection have been usually based on serological
methods, and RSV seropositivity has been reported to reach
73% in goat populations worldwide [15, 18, 27]. However,
only few studies have used immunohistochemistry for
evidencing RSV antigens [9, 24]. In the present study, the RSV
antigen could not be detected by immunohistochemistry in
any of the 105 cases with interstitial bronchopneumonia
and only 7 (6.67%) cases were determined positive by the
direct immunofluorescence test. In previous studies, it was
reported that positive cases could be evaluated as negative
because of the inactivation of immunogenic viral epitopes
in tissues fixed with formalin [6, 9, 13]. In this study, it was
considered that the inability to detect RSV positivity in
caprine lungs by immunohistochemistry may result from
fixation of samples with formalin. According to the results
of the present study, when difficulties in detection of specific
histological lesions in natural RSV pneumonias of goats were
taken into consideration, the DIF method was found to be a
more sensitive method compared to immunohistochemistry
for a definite diagnosis, despite difficulties in detailed
examinations of cellular structures in frozen sections. Thus, it
was concluded that the DIF method could be used as a more
appropriate method for a direct diagnosis of RSV infections
in goats.
As a conclusion, RSV antigens were found in the present
study at a rate of 6.67% using the DIF method in interstitial
bronchopneumonia cases in goats in the Elazığ region
and the role of this virus in the development of interstitial
bronchopneumonia in goats would be out of importance.
123
5.
6.
7.
8.
9.
10.
11.
12.
13.
References
1. ALANSARI H., POTGIETER L.N.: Molecular
cloning and sequence analysis of the phosphoprotein,
nucleocapsid protein, matrix protein and 22K (M2)
protein of the ovine respiratory syncytial virus. J. Gen.
Virol., 1994, 75, 3597-3601.
2. BAKER J., FREY M.: Bovine Respiratory Syncytial
Virus. In: Veterinary Clinics North America, BREEZE R.
(ed.): Veterinary Clinics North America. WB Saunder,
Philadephia, 1985, pp.: 275.
3. BELKNAP E.B., CISZEWSKI D.K., BAKER J.C.:
Experimental respiratory syncytial virus infection in
calves and lambs. J. Vet. Diagn. Invest., 1995, 7, 285-298.
4. BRYSON D.G., EVERMANN J.F., LIGGITT H.D.,
FOREYT W.J., BREEZE E.G.: Studies on the pathogenesis
Revue Méd. Vét., 2013, 164, 3, 120-124
14.
15.
16.
17.
and interspecies transmission of respiratory syncytial
virus isolated from sheep. Am. J. Vet. Res., 1988, 49,
1424-1430.
BUNT A.A., MILNE R.G., SAYAYA T., VERBEEK M.,
VETTEN H.J., WALSH J.A.: Paramyxoviridae. In: Virus
taxonomy, Eigth report of the International Committee
on Taxonomy of Viruses, FAUQUET C.M., MAYO
M.A., MANILOFF J., DESSELBERGER U. and BALL
L.A. (eds.), Elsevier, Academic Press, London, 2005, pp.:
655-671.
CASWELL J.L., WILLIAMS K.: The respiratory system.
In: Pathology of domestic animals, JUBB K.V.F.,
KENNEDY P.C. and PALMER N. (eds.), Saunders
Elsevier, Edinburgh, UK. 2007, pp.: 594-622.
DUNCAN R.B., POTGIETER L.N.D.: Antigenic
diversity of respiratory syncytial virus and its implication
for immunoprophylaxis in ruminants. Vet. Microbiol.,
1993, 37, 319-341.
ELERAKY N.Z., KANIA S.A., POTGIETER L.N.: The
ovine respiratory syncytial virus F gene sequence and
its diagnostic application. J. Vet. Diagn. Invest., 2001, 13,
455-461.
ELLEN B., BELKNAP E.B., CISZEWSKI D.K., BAKER
J.C.: Experimental respiratory syncytial virus infection
in calves and lambs. J. Vet. Diagn. İnvest., 1986, 7, 285298.
FORGHANİ B.: Diagnosis by viral antigen detection.
In: Lennette’s Laboratory diagnosis of viral infections,
JEROME K.R. (ed.), Informa Healthcare, New York and
London, 2010, pp.: 113-132.
GULBAHAR M.Y., CABALAR M., ERTURK
A.: Detection by immunoperoxidase tecnique of
parainfluenza type-3 virus and respiratory syncytial
virus antigens in naturally occuring pneumonia in
lambs. J. Fac. Vet. Med. Yuz. Yil., 2002, 13, 74-77.
GURCAY M., BOLAT Y.: Serological survey of
Respiratory Syncytial Virus (RSV) infections of sheep in
Elazig and it’s vicinity. Firat Uni. Vet. J. Health Sci., 1996,
10, 289-293.
HAINES D.M., CLARK E.G., CHELACK B.J.: The
detection of bovine respiratory syncytial virus in
formalin fixed bovine lung with commercially available
monoclonal antibodies and avidin biotin complex
immunohistochemistry. Can. J. Vet. Res., 1989, 53, 366368.
JACOBS J.W., EDINGTON N.: Experimental infection
of calves with respiratory syncytial virus. Res. Vet. Sci.,
1975, 18, 229-306.
JETTEUR P., THIRY E., PASTORET P.P.: Serological
survey of IBR, CHV-2, PI-3, bovine respiratory syncytial
virus and rinderpest virus in sheep and goats in Zaire.
Rev. Elev. Med. Vet. Pays. Trop., 1990, 43, 435-437.
KAHRS R.F.: Parainfluenza 3. In: Viral disease of cattle,
KAHRS R.F. (ed.), Iowa State University Press, Ames,
1981, pp.: 171-181.
LEHMKUHL H.D., SMITH M.H., CUTLIP R.C.:
Morphogenesis and ultrastructure of caprine respiratory
syncytial virus. Arch. Virol., 1980, 65, 269-276.
124
18. MALLIPEDDI S.K., SAMAL S.K.: Analysis of the ovine
respiratory syncytial virus (RSV). J. Gen. Virol., 1993, 74,
2787-2791.
19. MASOT A.J., GOMEZ L., MARTÎNEZ M.S., GAZQUEZ
A., REDONDO E.: Experimental infection of lambs
with bovine respiratory syncytial virus : pathological
and haematological studies. Rev. Méd. Vét., 1993, 144,
773-780.
20. PAGE W.G., GREEN R.G.: An improved strain for
distemper inclusions. Cornell Vet. 1942, 77, 265-268.
21. SHARP J.M., NETTLETTON P.F.: 2007. Acute
respiratory virus infections. In: Diseases of sheep,
AITKEN I.D. (ed.), Blackwell Publishing, Edinburg,
2007, pp.: 207-208.
22. SMITH M.H., LEHMKUHL H.D., PHILLIPS S.M.I.:
Isolation and characterization of respiratory syncytial
virus from goats. Am. Assoc. Vet. Lab. Diagn., 1979, 22,
259-269.
CERIBASI (A. O.) AND COLLABORATORS
23. STOTT E.J., TAYLOR G.: Respiratory Syncytial Virus.
Arch. Virol., 1985, 84, 1-52.
24. TRAHMAN T., SINGH B.: Incidence and pathology of
viral pneumonia in goats. Ind. J. Anim. Sci., 1990, 60,
1159-1162.
25. VAN DER POEL W.H.M., LANGEDIJK J.P.M., KRAMPS
J.A., MIDDEL W.J.G., BRAND A., VAN OIRSCHOT
J.T.: Bovine respiratory syncytial virus antibodies in
non-bovine species. Arch. Virol., 1995, 140, 1549-1555.
26. WELLEMANS G.: Bovine Respiratory Syncytial Virus.
In: Virus infections of vertebrates, HORZIENK M.C.
(ed.), Amsterdam, Oxford, New York, Tokyo, 1990, pp.:
363-372.
27. YESILBAG K., GUNGOR B.: Antibody prevalence
aganist respiratory viruses in sheep and goats in NorthWestern Turkey. Trop. Anim. Health Prod., 2009, 41,
421-425.
Revue Méd. Vét., 2013, 164, 3, 120-124