A. tJ. Veı. Fak. Derg. 35 (2-31: 289-298. 1988
DIAGNOSIS OF INFECTlOUS BCRSAL DISEASE (ıBO) BY IMMLNOFLlJORESCENCE
Rıfkı Hazıroğlu' Makoto HaritanP
Minoru Maeda2 Kikuyasu Nakamura2
Minoıu Nadta3 Infcksi)'öz bursal hastalığının immıınofloresans ilc teshisi
Özet: Beş hafialık SPF piliçlerde infeksiyöz bursal hastalık de-neyselalarak çalışılııııştır. İnfeksiyliz bursal hastaltk virusıı ile eııfekte piliçleriıı leııfokl ve diğer organları histopatolojik olarak incelenmiş
ve belirgiıı lezyonlara bursa Fabricius'ta rastlanmıştır. Daha az
ııek-roza rastlanan diğer lenfoid organlann yüksek rejenerat(f potansiyele
salııı) olduğu glizlenmiştir. Karaciğer ve böhreklerde nonspesifik ve
hafil patolojik değişiklikler tesbit edilmiştir. Bursa Fahrieius'un doıı-durma kesitleri ve formaliııde tesbit edilmiş parafin kesitlerinde immu-nofloresans tekniği kul/amlarak infeksiyöz bursal hastaltğı virusu tesbit edilmiştir. Rutin teşhis için formalinde tesbit edilmiş parafın kesitlerinin de immuııofloresans tekniği ile viral ant(jenin saptal1l11asll1dauygıııı olacağı ı'e bu tekniğin patoloji laboratuvarıanna yararları aÇlklanmışftr.
SummaQ': Infeetious bursal disease in 5-lI'eek-old Speeifie
Patlıo-gen Free C1ıickens was experimental/y studied. Histopathologie
exami-nation of Iymphoid and ııonlymphoid tissues from infectious bursal disease virus (I BD V) i4ected C1lickens confirmed that the predol11inant lesion was IYl11pllOidf1(icrosis iıı the bursa of Fabricius (BF). Other Iymphoid organs were much less severely aj/ected and had greater regenerarive
potential. Nonspecifie and relatively mild changes were defiııed in the
/iver and kidııeys. Immunof/uorescent tecl1l7ique was used to detect in-fectious bursal disease virus in formalin-fixed paraffin-embedded tissues
i University of Ankara, Faculty of Vetcrinary Medicine, Department of Patlıüioj!Y, 061LO Dışkapı IAnkara - TURKEY.
2 Poultry Disease Laboratory, National Institute of Animal Health, Kurachi, Seki, Gifu 501-32 JAPAN.
3 Diagnostic Patlıology Laboratory, National Institute of Animal Health, Yatabe, Tsukuba 305, JAPAN.
290 R. Hazıro~lu - M. Maeda - K. Nakamura - M. Hariıanİ - M. Nariıa
andfrozen sections of the bursa of Fabricius. For rouline diagnostic pur-poses, viral antigens can be detected using immul1ofluorescence the be-nefit of which has been described in the pathology laboratory.
Introduction
Infectious bursal disease (ıBO), an acute viral disease of young chickens, was first described by Cosgrove in 1962 (3). The disease is widespread in chickens and is of great economic importance for both broiler and pullet growers (3, 5, 7, 8,
ıo).
Infectious bursal disease is normal!y diagnosed in the veterinary laboratory by the isolation of the causative virus in eggs and for cel! cultures and the demonstration of bursal lesions. These procedures, however, can be relatively time consuming. The use of direct electron microscopic examination of faeccs (12) and bursal suspensions (i i) as a possible method for the rapid diagnosis of ıBO has been described. Several investigators have reported detection of various sort of viru-ses in formalin-fixed paraffin-embedded tissues (6, 14, i5, 17). Infec-tious bursal disease virus (IBDV) has been demonstrated in frozen (2, 4, 9,
ıo,
i3) and fixed parafin-embedded sections (9) and impression smears made from the bursa of Fabricius (BF) (i).This report describes the detection of IBDV in frozen and for-malin-fixed paraffin-embedded tissııses using the direct fluorescent antibody technique.
Materials And Methods
Virus: The vacuum dried Jl strain of IBDV, kindly provided by
Dr. Hihara (Poultry Disease Laboratory of National Institute of Ani-mal Health, Gifu. Japan) was added to 2 ml minimum essential medium (MEM),
%
5 fetal bovine serum, and the usual antibiotics and used as an inoculum suspension for preliminary examination. Four chickens were inoculated intra-orally with 0,2 ml viral suspension. Two chickens were killed at 3 days postinoculation and the BF were collected. The collected BF were mixed with MEM five times ıısing a special tube, centrifuged at 1000 rpm for i5 min. and filtered using a 450 nm pro-posal filter and the col!ected supernatants were used as inoculum sus-pension. The remaining two chickens were killed at 21 day s postino-eulation and bled for serum collection.lNfEKSİvöz BURSAL HASTALIGININ TEŞHiSi
Experimenta/ design: Chickens were obtained from a
spccific-pathogen-free white leghorn flock (POL-I) at 5 weeks of age and
maintained at the Poultry Disease Laboratory of the National Insti-tute of Animal Health, Gifu, Japan. Twenty ehiekens were inoculated intra-oraııy with bursal suspension of IBOV. Four chiekens per aday were kiııed on day s 3, 4, 5, 6 and \ 4 after inoeulation. Then the BF, spleen, gut, caecal tonsil, kidney, liver, lung and Harderian glands were
removed. Uninoculated six control chickens were separated from
!BOV infected chickens. On days 3, 5 and 14 after inoeulation, two control chickens were kiııed and same organ material colleeted. Chic-kens were examined daily for clinical signs of ıBO. Aıı chicChic-kens were offered food and water ad libidum.
Immul1oj/uorescence: Fluorescein-Conjugated anti-IBOV chicken
gamma globulin was prepared by a method described earlier (\ 6). Frozen samples of BF were cut 8tL thick İn a eryostat microtomc. The sections were fixed in cold acetone for \ O min., air dried, and dipped in phosphate buffered saline (PBS). The sections were stained with fluo-rescein isothiocyanate-conjugated antiserum (FICA) to the J i strain of IBDV for 30 min. in a moist chamber at 37
"c.
After threefold washed in PBS, 5 min. each time, and a brief rinsed in distiııed water, the sections were mounted on s\ides using 50%
glycerol-PBS solution. Forma\in-fixed paraffin-embedded sections of BF were prepared. De-paraffinisation was made with xylol and grade series akoho\. Tissue sections were thoroughly rinsed in PBS, then sections of RF werepre-treated with 0,1
%
pronase (Kaken K.K., Tokyo-Japan) in PBS, pH7.2 for 5 min. at 37 oC, then rinsed in PBS for 5 minutes. Af ter that the sections were ineubated with FICA for 30 min. in a moİst chamber at
37 oC, then threefold washed in PBS, 5 min. each time, and CL brief
rinsed in distilled water, mounted on slides and observed with a fluorescence microscope (Nikon, Fluophot, Microphot V series, Japan).
Liglıt microscopy: Tissue samples were fixed in LO
%
neutra\buf-fered formalin and embedded in paraffin wax and stained with hema-toxylin and eosin (HE).
Results
Gross and microscopic lesions oj lymplıoid organs: No significant
gross lesions were observed in orgaris except BF. The chief and c:onsis-tent finding produced was an initial increase in the weight of BF (Tab-le 1). This was dear by the 3 rd postinoeulation day. By the 5 th
pos-Table i. Data of the ehiekens med in the study
i
Days
Post-BF Body Relative
inoeulation
Purpose Sex (DPI) weight (Gr) weight (Gr) wei~ht
i
For inGı:u!um Femalc 3 1.90 368 0.51___ o .---"-'--- ---_... ---
--suspension Female :i ı.30 360 0.36 Female Control 1.20 494 0.24 CONTROL -- -._- ----.-- ----_.--
. ---Female Control 1.00 475 0.21 Male 3 1.60 590 0.27 ---- ----._---- --_._- --- - ---_.-Male 3 1.30 5H 0.22 Experiment --- ---_.._-
--- --_._--- --,._-- --Male 3 1.40 484 0.28 ---- --_._-._". --- -_.-._----_._---i
MaleMale :i4 1.101.20 482505 0.240.21i
--,-- --.- ---- -._---- -. ._---Male 4 0.60. 5ıS O. II Expı~rimcııt ---- ---.---- -_._--- .-_
.. _--Male 4 0.70 :'42 O.ıı ._._---"--- .._-_.- -- --- ---Female 4 1.00 440 0.22-i
Fcmalc Control 1.80 582 0.30 CONTROL ._--_.. --_._..- --- --- - ----Female Control 1.70 486 0.34i
Female 5 0.60 464 0.12 -."-- --- __ o ._-_._--- ._---Male 5 0.90 556 0.16'i
Experiment --- ---,---. --- --.._---- ---Female 5 0.60 466 0.12 - .._-
--_._---- ._--- --- ----Fcmale 5 0.60 464 0.12 Female 6 0.70 488 0.14 --- ---"--- - --__ o ---Female 6 060 568 0.10 Experiment ___ o ._---- --- --- ---Femalc 6 0.60 566 0.10 --- --- -- -"._---- -._--Male 6 0.70 524 0.13-Female Control 1.40 638
o.ıı
CONTROL ---- --- ._---Female Contwl 1.20 592 0.20 Feınale 14 0.55 693 0.07 ---- -_._--- --- -- ---Female J4 0.40 662 0.06 E~.perimer:t .----_ ..-,._._--- ---- ----- _.. _----Female J4 0.70 617 O.Ji ---- ---_. ---
-_
.._--- _._--Male 14 0.55 608 0.08 Female 21 0.50 658 0.07 For serum -- -_.'._--eollection Fcmale 21 0.30 522 0.05Relative weight bursa of Fabrieius weight body weight x 100
iNfEKSiYÖZ BURSAL HASTALIGININ TEŞHİsİ
tinoculation day, one gained the impression that the BF was smailer than in the controls, and impression whieh was easily confirmed by the 14 th postinoculation day (Fig. i). Typical microscopic lesions of ıBO were observed in BF of chickens when examined on the 4 th pos-tinoculation day (Fig. 2). The changes had sprcad to all Iymphoid fol-licles in the BF. As well as this, large globules of lipids werc present in all cell type within the bursa, including the interfollicular connective tissue cells.
fig ı. Bursae of f2bricius 14 days posıinoculatioıı wiıh lBDV. Top 2bursae wcrc from unİnoculated conlrols; lower 4, İnfccted. l'\ote the atrophy.
The spleens from chickens killed on days 3, 4, 5 and 6 after ino-culation, contained focal areas of necrosis of Iymphoid nodules and periarteriolar Iymphoid sheats. The spleens appeared normal at 14 days postinoculation, except for İncreased Iymphoid foci.
The ceacal tonsils of IBOV infected chickens exhibited some
Iymphoid ceU loss from the Iymphoid nodules, with macrophage in-filtration and an occasional crypts containing necrotic material and heterophils. No lesions were noticed in any samples from unİnoculated controls.
The thymus was difficult to evaluate. But after the 4 th day pos-tinoculation the cortex of so-called thymocytes appeared to thin out, which persisted through the 14 th day postinoculation.
...
29,1. R. Hazıroğıu - M. Maeda - K. Nakaınura - M. Haritanı - M. Narİta
Fig 2. The bursa of Fabrİeius of a ehicken infected 4 days preyiously. In addition to the degenerative process of t!ıe follie!cs, interfollicular spaees are increased and
edenıatous. HE x 120
Nonspeeifie and mild changes were observed in the liver and
kidneys.
Imli111l1o[luorescCllce: The detection of viral antigens due to eon-jugate antiviral globulin, as revealed by speeifie f1uoreseenee, was seen throughout affeeted follicles in both of the frozen and fixed paraffin-embedded seetions (Fig. 3 and 4) whereas speeifie f1uoreseenee was not observed in uninfeeted control s ehiekens. Bursal tissue examined 3,
4, 5 and 6 days after inoeulation had widespread eytoplasmie f1uores-eenee believed to be in the maerophages. Fluorescing eells were pre-sent 14 days af ter inoeulation, but less than on 3,4, 5 and 6 days after inoeulation.
Nonspeeifie, yellow f1uoreseenee was aeeepted to be eaused by lipids globules, fat eells and some lipid containing eytoplasmie globu-les. Orange color f1uoreseence was emitted by eosinophils.
iNFEKSivöz BURSAL HASTAllGININ TEŞHisi 295
Fig 3. Frozen section of bursa of Fabrieius fronı a ehieken infeeted with IBDV, killed 6 days arter inoeulation and stained wiıh IBDV eon.iugate.
Fig 4. Formalin-fixed paraffin-enıbedded section of bursa of Fabrieius from a ehickcn infeeted with IBDV, killed 5 days after inoeulation and stained with IBDV
~9(1 R. Hazıroğlu - M. Macda - K. Nakamura - M. Harilaııi - M. Narila
Two-step blocking and absorbtion tests were performed for.chec-king the specificity of the J i conjugate in frozen sections of affected bursae. No immunofluorescence was detected in any of these tests using the Jiconjugate.
Discussion and Conclusion
Histopathologic cxamination of Iymphoid and nonlymphoid tissues from chickens infected with IBDV confirmed that the predo-minant lesions were see n in the RF. Bursal pathology was characterized by acutc Iymphoid necrosis. lo. follicles with moderate changes, some amount of Iymphoid regeneration towardnormal bursal architecture was evident day 14 postinoculation. Necrosis in nonbursal Iymphoid organs (spIeen, thymus, ceacal tonsils) was much less severe. All of these results are consistent with previous observations (2, 4, 5, 7, JÖ,
18).
The use of immunofluorescence for rapid diagnosis of viral in-fections in animals is well known (I, 5, 8). J mmunofluorescence, as a method for rapid diagnosis of iBDV infections, is less widely used in avian pathology laboratories. lmmunohistological examinations of viral antigens have usually been carried out on frozen sections. Routine surgical material, however, is almost invariably received iiı formalin or has been previously fixed and embedded in paraffin wax. In these circumstances the requirement for an immunological studyonly be-com es apparent after microscopic examination of the paraffin sectİons.
lt is generally believed that formalin-fixed tissues are unsuitable for immunologic studies as antigen and antibody complex formation is inactivated by conventional histological methods. Recently several investigators have successfully applied the immunofluorescent antibody technique to staining varİous antigem in formalin-fixed, parafin-em-bedded tissues af ter trypsinizing treated tissues (6,14,15,17). Jönsson and Engström (1986) have achieved the detection of IBDV using im-munofluorescent technique in fixed paraffin-embedded material (9). They have used various type offixative solutions and different digestion procedures whereas, in our cases, wc have used only neutral buffered formalin for fixation and O, i
%
pronase for digestion. They have also used pronase digestion for 15min. at 37oc.
We used pronase digestion for 5 min. at 37oc.
Also this procedure gave good result5.The decision as to which test to use for the diagnosis of lBDV infection will depend on the facilities available. The results of
experi-iNFEKSiYÖZ BURSAL HASTAllGININ TEŞHiSi ~97
ments have shown that iınmıınoflııoreseenee on frozen seetİons of BF is preferred if possible. it can be performed very rapidly, a large number of specimens can be processed quickly and it can deteet the viral antigen. If fresh material is not available, formalin-fixed paraffin-embedded material is alsa convenient for the detcetian of lSDV.
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