A. (J. Veteriner Fakültesi Bakteriyoloji ve Salgılar Kürsüsü Prof Dr. Ömer Ertürk
T. B. Etlik Bakteriyoloji ve Seroloji Enstitüsü Dr. Ahmet Özsoy
AN OUTBREAK OF EQUINE VIRUS AB OR TION IN TURKEY
I. ISOLATION AND IDENTIFICATION OF
RHINOPNEUMO-NlTIS VIRUS IN CELL CULTURES
Hasan Başkaya
*
Mesadet Doğuer**
Salih Yılmaz* *
Hamza Keskintepe* Bekirt
yigören* *
Ali Demir**During the last two decades it has been established that the the disease formerly described as equine influenza was not a single entity. There are, however, three distinct virus diseases which may have similar symptoms and similar complications, and each of which may be fitted into the complex of respiratory disorders of horses. These diseases are equine rhinopncumonitis, viral arteritis of horses and a true equine influenza caused by a virus typical of the myxovirus group (5, 7).
Dimock and Edwards (I, 2) was the first to describe the equine virus abortion and to determine its filterable etiology. The results of investigation conducted by Manninger (II), Manninger and Cson-tos (12) led them to decide that virus abortion ofpregnant mares was caused by equine influenza virus. Other studies (7) revealed that equine influenza virus was identical with the equine abortion virus. Subsequent studies by DoU et aL. (5) established that the disease was primarily a respiratory infection with abortion occuring as a result of infection in pregnant mares. They named the disease equine rhino pneumonitis and the agent equinc rhinopneumonitis virus.
* From the Dept. of Bacteriology, Faculty of Veterinary Medicine, University of Ankara, Turkey.
310 H. Başkaya ve Arkaddşlar!
The initial researeh on the rhinopneumonitis virus abortion problem was direeted toward finding a suitable teehnique that the virus eouldbe intr0dueed direetly into the fetus earried by pregnant mares, and that 100per cent offetuses so inoeulated would be aborted
(4). This proeedure enabled laboratory workers to maintain and to identify strains of virus. The first sueeess in the basic laboratory studies wasdevelopment of a eontplement fixation test for the virus(8). Through this test, it was possible to study the prevalenee and spread of the disease, and to learn that on nearly aıı farms almost every hor-se was infeete.d prior to oeeurrenee of the first abortion.
Doıı et aL. (9) was the first to propagate the virus in suekling Syrian hamsters. The hamster-virus system has providcd a sensitiye laboratory tool for virus neutralization tests whieh serve for speeifi-eaııy identifying the virus (6). Soon there after, hamster-propagated abortion viruses were eultivated on the ehorioaııantoie membrane of the ehieken embryo (I o).
Sinee 1953, various eell eultures were investigated for their eapa-city to support propagation of the rhinopneumonitis virus (I 5, 16,
i7, 18).
Shimizu et aL.(20, 2i) reported on the use of horse kidney
eul-turc system in the isolation of virus from natural eases of equine abor-tion. MeCoııum et aL. (I 3) described the isolation and propagation of rhinopneumonitis virus in primary monolayer eeıı eultures of eq-uine, ovine and porcine kidney.
The etiologieal agent of equine abortion in Turkey has been, for a long time, considered to be Salmonella abortus equi. But in those of aıı baeteriologieally negative eases the eause was unknown and remained to be sought. However reeent study (3) suggested that viral abortion whieh oeeurs in most of the horse-rasing regions of the world was also prevalent in Turkey. The subsequent histopat-hologieal studies by Pamukçu ct aL. (I 4) definitely revealed the pre-sence, in Turkey, of equine abortion due to rhinopneumonitis virus by demonstrating speeifie intranuclear inclusion bodies in various speeimens of tissues obtained from aborted fetuses.
So far, there have bcen no isolations of etiologie agents of eq-uine abortion virus in Turkey. This report describes the isolation and identifieation of the virus in primary monolayer eulture of kidney eeııs derived from various ages of sheep, and eompares its immunolo-gieal relationship to the equine rhinopneumonitis virus as weıı as
An Ouıbreak Of Eoume Vırus Abortıon ın Turkey
Meterials and Methods
311
Specimens of liver, lungand spleen were harvested with sterile precautions from five naturally aborted fetuses. Portions of each organ dispensed into vials, and ston~d at -200C. Vials of tissue were
discarded after being thawed once. To prepare tussue suspensions for the biologic test, frozen tisses were thawed slowly in a refrigerator at
+
4 oc., minced with sterile scissors and grinded in a ultra - mi-xer. Earle's balanced salt solution was used as a diluent to give aio per cent crude suspension of the tissue by weight. This suspension
was centrifuged at 3000 rpm for 5 minutes. The supernate was the inoculum. 2000 units of penicillin and 2 mgr. of streptomycin were added to each mL. of the inoculum.
Cell Culll/re.
Primary monolayer culture of kidney cells prepared from vari-.ous ages of sheep were employed in the present study. Dispersed cclls were obtained by digesting tissue fragments of the kidney cor-tex with trypsin as decribed by Youngner (22). The growth medium employed consists of Hanks' balanced salt solution, containing 0.5 per cent lactoalbumine hydrolysate, io per cent calf serum
inac-tivated by he:ıting at 56°c. for 3° minutes after filtration through a Seitz EK disc. 100 units of penicillin and o.i mgr. of streptomycin
per mL. The maintcnance medium used was the same. One milli-liter of 0.5 per cent eell suspension prepared was used for standard culture tubc, and 15 mL. for a milk dilution bottle. İncubatian was carried out without motion at 37°c. Botdes were laid flat in trays whilc tubes were inclined at a slight angle. Cultures were Ieft undisturbed for 48 hours, after which time they were usuaIIy stuck to the glass and beginning to multiply. The old growth medium was always replaced after 48 hours. Subsequent changes ofmedium we-re at intervals of 3 to 4 days. Excelent cell sheets wewe-re produced af-ter 5 days of incubation and maintained in good condition for at least 8-10. days.
For virus isolation, the growth medium was remove d from milk dilution bottles, and 1.5 mL. of the specimen was inoculated. Mter incubatian at room temperature for 2 minutes, i3.5 mL. of growth
medium was added into the botdes and left at 37°c. for 2 hours. At the end of this period, fluids were removed from batdes and after adding i5 mL. of fresh medium the cell cultures were incubated at
312 H. Başkaya ve Arkadaşları
i
L
37°e., and examined daily under a mieroseope for eytopathie effeets. At the termination of trial period, the eulture fluids and eeııs were harvested and either inoeulated immediately or frozen.
Equine Rhinopneumonitis Virus.
RAC-H strain was obtained through the eourtesy of Prof. Dr. V. A. Mayr, Germany. Thİs eell eulture adapted strain was passed in monolayer eulture of sheep kİdney eeııs at our laboratory, and the virus at the IIth passage was employed as seed virus İn the present study.
Antisera.
Antisera against the RAC-H, and one of the our isoiate was pre-pared in rabbits. Cell eulture of sheep kidney İn milk dilution bottles wcre infeeted with RAC-H and our isolate. When the eytopathie effeet was eomplete, the whole eulture was homogenized and eent-rİfuged at 3000 rpm for ıo mİnutes. The supematant fluid was employed as viral antİgen for immunization of rabbits. Animals were gi -ven 5 intra-venous injeetions with RAC-H (ı07 TCID 50)' and our isoiate (ıosTCIDso) at intervals of 3 days. The amounts admİnistered were 0.3 m!., 0.5 m!., ı.o m!. ı.S m!., and 2.0 m!. The rabbits were bled to death on the sİxth day after the last injeetion. Sera were se-parated and stored frozen at - 200c.
Neutraliz.ation Test.
A stoek of the isolated virus was prepared in eell eultures cu 1-tivated in milk dilution bottles. After 3 days of ineubation virus growth was eomplete as indieated by eytopathogenie effeets, and the suspending medium and eeııs was removed from several bottles and pooled. From this homogenized whole eulture the ecllular debris was removed by eentrifugation at 3000 rpm for 20 mİnutes, and the dear supematant dİstributed İn ampoules and Iyophilized.
For vrius titration, the virus material in an ampoule was diluted with Hanks'solution to make serial tenfold dilutions, and o. ı m!. of eaeh of the dilutions was inoeulated into groups of 4- tube eultures per dilution. The inoeulated tu bes were ineubated at 37°e. for one week. TCID50 titre was eakulated by the Reed and M ueneh method (ı 9) based on the developing eytopathie effeets.
Serum was inaetivated by heating at S6°c. for 30 minutes, and it was then diluted in twofold steps with Hanks' solution starting with a dilution of ı :2. The serum-virus neurtralization tests were set
An Outbreak Of Eoume Vırus Abortlon ın Turkey 313
up by mixing one mL. ofeaeh serum dilution with one mL. of virus sus-pension containing 100 TCIDso per mL. The mixtures left at room
temparature for one hour, and eaeh of the serum-virus mixture was then seeded into 4 tubes of eeıı eulture with o.i mL. amounts. 0.9 mL.
of maintenanee medium was added to eaeh tube and ineubated at 37°C. The results of neutralization trials was reeorded on the bascs ofeytopathie effeets. Complete neutralization of virus in 4 tubes was aeeepted ast he titration end point.
Results
Virus Isolation.
Attempts were made to isoiate the etiologieal agent of equine abortion in primary monolayer eultures of ovine kidney eeııs. Speei-mens of liver, lung and spleen were eoııeeted from 5 aborted fetuses. The first isolation of virus was obtained from a fetal liver. Baeterio-logieal tests failed to reve al any pathogens in these speeimens of organ'>. Six milk dilution bottles of monolayer eeıı sheets were inoeu-lated with aı: io suspension of liver tissue in Earle's solution. Among
these eeıı eultures only three ofthem showed obvious foci eytopathie change whieh began 36 hours after inoeuation , and affeeted the whole eeıı sheet at 3 - 4 days. İn the remaining eell eUltures the ey-eytopathie changes wc re deteeted 72 hours foııowing inoeulation. İnfeeted eultures were harvested at 5th dayand transfered in fresh monolayer eulture of kidney eeııs. İn the second and later passages the eytopathogenie effeets were observed in aıı İnoeulated of eell eultures, 36 hours after inoeulation. The infeeted eell eultures eom-pared with uninfeeted ones, and the control eultures were found to be unaffeeted during the trial period. The first eytopathie effeets appcared in seattered areas as rounded eeııs and the number of such eeııs increased rapidly affeeting adjoining eeııs. By the 5th to 6th day the degeneratd eeııs aggregated and were faııing off the glass.
Neutralization . Test.
The 4 isolates whieh were reeovered from eases of the same out-break found to be strains of a single virus. Beeause eytopathogenie changes developed in ovine kidncy eell eultures were indistinguishable from eaeh other, and all of the 4 isolated viruses weıe well neutralized by the anti-serum produced against one of them to a similar cxtent. The antigenie property of the isolated viruses was investigatcd by neutıalization test employing rabbit antiserum produeed against
314 ır. Başkaya ve Arkadaşları
the RAC-H strain and one of the newisolatc. The anti-RAC.H serum neutralized aıı of the 4 agents to a lesser extent, but it neutralized well the homologous virus.
İn view of the serum-neutralization test result, along with their eytopathogenieity in eell eulture, it was eoncluded that the agents were strains of rhinopneumonitis virus. The presenee of antigenie I'elation between isolated viruses and the RAC-H strain was also de-termined.
Lyophilized eulture fluid of one our isoiate was sent to Germany for type determination. This virus strain was identified as "equine rlıinopneumonitis subtype 2" by Prof. V. A. Mayr, Faeulty
ofVeteri-nary Medicine, Munieh. .
Discussion
Various eell eulture systems have been investigated for the iso-lation and propagation of rhinopneumonitis virus by many workers (13, 15, 16, 17, ı8, 20, 21). Reeent work on an outbreak of abortion in mares, in Turkey: has revealed the presenee of speeifie eosinophilie intranuclear inclusion bodies in speeimens of tussues of aborted fe-tuses. These findings were pathognomie for virus abortion (14).
The present study has shown that the virus recovered from an outbreak of abortion oeeurred in a government stud in Turkey can be easily grown in primary monolayer eultures of ovine kidney eeııs. This eonfirms the results of other investigators (13, 20). Serial passages were readily aeeomplished in this eeıı eulture. Definite eytopathie effeets was evident 36 hours foııowing inoeulation. The 4 isolated agents considered to be strains ofa singlc virus. Beeause they were isolated from eases of a single outbreak, their eytopathogenie effeet in eulture of ovine kidney eeııs was indistinguishable from eaeh other, and in the eross-neutralization test no differenee was demonstrated am-ong them. On the other hand, the eross-neutralization test revealcd a distinetion between the German RAC-H strain and the newly isola-ted virus strain.
One of the Iyophilized strain was sent to Germany, and typed as "rhinopneumoni tis su btype 2".
This is the first to report the isolation of the rhinopneumonitis virus from an autbreak of abortion in Turkey.
,--- -
---_._---An Outbreak Of Eoumc Vınıs AbortlOn m Turkey
Suınınary
315
An outbreak of abortion oceurred among mares in Karacabey stud in Turkey during the 1966 foaling season. 4 eytopathogenie viral agents were isolated from speeimens of 5 aborted fetuses in primary monolayer eulture of ovine kidney eclis. The isolated strains were found to multiply and produee cytopathic changes in this ccll cul-turc. Serial passages were readily aceomplised. The 4 solated agents considered to be strains of a single virus because they were recovered hom cases of a single outbreak, their cytopathogenieity in eeU cu 1-tures was indistinguishable from each other, and in the erossneut-ralization test no difference was demonstrated among them . However, the cross neutralization test revealed a difference between the Ger-man RAC-H strain and the newly isolated strains.
The isolated agents were identified as equine rhinopneumonitis virus, and one of the lyophilized strain \Vas sent to Germany and typed as - "rhinopneumonitis subtype 2".
Türkçe Özet
i966 yılıdoğum mevsiminde Karacabey Harası kısraklarında
görülen yavru atma olaylarından 4 adet sitopatojenik virus izole edil-miştir. !zalasyonda koyun böbreği hücre kültürleri kullanıldı. İzole edilmiş suşların seri halindeki pasajiarına bu hücre kültürlerinde mu-vaffak olunmuştur. Bu etkenlerin, koyun böbreği hücre kültürlerinde husule getirdikleri sitopatolojik değişikliklerin birbirine benzemesi, izole suşlardan birine karşı hazırlınmış immun serumla hepsinin nötrali-ze olmaları ve aynı salgın olaylarından izole edilmeleri sebebiyle, aynı virusun suşları oldukları kanaatine varıldı. Çapraz nötralizasyon testi Almanya orijinli RAC-H suşu ile yeni izole edilmiş yerli suşlar arasında antijenik bir fark göstermiştir. Yerli suşlardan biri si liyofilize edilerek Almanya'ya gönderilmiş ve Hannovel' Yüksek Veteriner Okulunda ve Münih Veteriner Fakültesinde "rhinopneumonitis subtype 2"
olduğu tesbit edilmiştir.
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316 H. Bdşkaya ve Arkadaşları
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