- Email: [email protected]
Failure to demonstrate transplacental transmission of parainfluenza-3 virus by inoculation of pregnant immune sheep
THERIOGENOLOGY
FAILURE TO DEMONSTRATE TRANSPLACENTAL TRANSMISSION OF PARAINFLUENEA-3 VIRUS BY INOCULATION OF PREGNANT IMMUNE SHEEP Robert B. Grieve and Brinton L. Swift Division of Microbiology and Veterinary Medicine University of Wyoming, Laramie, Wyoming 82071 Reoeived for plblication Arqust 8, 1974 ABSTRACT Seven ewes with circulating PI-3 serum neutralizing (SN) antibody were divided into 3 groups. Ewes in Group 1 which consisted of 4 ewes, were inoculated intratracheslly, at midgestation with parainfluenza-3 (PI-3) virus isolated from an aborted bovine fetus. Four fetal lambs were removed surgically from 2 ewes in Group 1. These lambs were negative for precolostral PI-3 SN antibody and virus was not recovered from the tissues of the lambs. Two ewes in Group 1 did not deliver lambs. All ewes in Group 1 developed an increase in PI-3 SN antibody titers following inoculation. Two fetuses of the ewe in Group 2 were inoculated in utero-with PI-3 virus. Both lambs were stillborn and had elevated _precolostral PI-3 SN antibody titers. Virus was recovered from the tissues of 1 lamb. Group 3 consisted of 2 uninoculated ewes. All lambs born in Group 3 had negative precolostral PI-3 SN antibody titers and virus was not isolated from the fetal tissues. Pregnant immune sheep inoculated with virulent PI-3 virus resist transplacental transmission of virus. INTRODUCTION Parainfluenza-3 (PI-3) virus infections in sheep are common (1). The virus has been isolated from aborted bovine and ovine fetuses (2,3, 4), but the role of the PI-3 virus in fetal disease and abortion in ewes is unknown. Fetal disease and abortion have been demonstrated by intrauterine inoculations of virus in cattle and sheep (2,4,5). Pregnant immune heifers were found to resist transplacental transmission of PI-3 virus by parenteral inoculation with PI-3 virus recovered from an aborted bovine fetus (7). Experimental abortion has been reported in sheep by intravenous inoculation with PI-3 virus (2). This experiment was designed to study the possibility of transplacental transmission of PI-3 virus in pregnant immune ewes by intratracheal inoculation of the virus. PI-3 seronegative sheep were not available for this study. MATERIALS AND METRODS Experimental sheep. Seven aged, well nourished, western whitefaced ewes B the University of Wyoming Livestock Experimental Farm at Laramie, Wyoming. All ewes had existing PI-3 serum neutralizing (SN) antibody titers before experimental inoculation (Table 1). Rams fitted with marking harnesses were joined with the ewes for 54 days and colors on the harnesses were changed every 14 days. The ewes were con-
JULY-AUGUST
1974
VOL. 2 NO. 1.2
THERIOGENOLOGY sidered pregnant when they no longer were mounted and marked by the rams. The breeding dates were recorded and the ewes were moved to the University of Wyoming Veterinary Medical Research Center at Laramie after breeding. They were fed dehydrated alfalfa pellets daily with salt and heated water ad libitum. The ewes were separated into 3 groups according to their SN antibody titers, their breeding dates and route of inoculation. Experimental design. Group 1 (Table 1) consisted of 4 ewes, 3 of which had existing PI-3 SN antibody titers of 1:16 and 1 of 1:32. These ewes were inoculated intratracheally. Group 2 consisted of 1 ewe which had an existing PI-3 SN antibody titer of 1:32. A midline laparotomy was performed on this ewe for the purpose of fetal inoculation on the 66 day of gestation. This route of inoculation was used as a positive control to determine the response of the ovine fetus to PI-3 virus by direct inoculation. Group 3 consisted of two ewes with PI-3 SN antibody titers of 1:16 and 1:32. These ewes served as uninoculated controls. Virus. The virus was recovered from an aborted bovine fetus (4) and was previously characterized (6). The ewes in Group 1 were inoculated intratracheally with 10 ml of the isolate from bovine embryonic kidney cell cultures (BEK) containing lo5 TCIU50/ml of virus. The two fetuses in Group 2 were inoculated with 1 ml each of the same inoculum. Surgical procedures. A midline laparotomy was performed on the ewe.in Group 2 for an intrauterine fetal inoculation and on each ewe in Group 1 to recover the fetuses prior to the expected time of parturition. One million units of penicillin and 1.25.grams streptomycin were administered intramuscularly daily for four days after surgery. Clinical observations. Rectal termperatures of ewes in Groups 1 and 3 were determined every 3 days beginning 6 days before inoculation and continuing 17 days following inoculation and again at 23 and 30 days following inoculation. Blood was collected at the same time in a vacuum tube containing ethylenediametetraacetic acid for differential and total white blood cell (WBC) counts. Blood samples were collected 6 days before inoculation and 14, 30, and 51 days after inoculation from all ewes in Groups 1 and 3 for PI-3 serology. Blood samples were collected from the ewe..inGroup 2 one day before inoculation and 6, 37, and 69 days after inoculation. The ewes were observed daily throughout the gestation period. Clinical pathology. Total white blood cell (WBC) counts and differential WBC counts were determined from blood samples collected from ewes in Groups 1 and 3. A hemacytometer was used to determine total WBC counts and blood smears were stained with Giemsa stain to determine the differential WBC counts. Serological examination. Blood samples were collected for serological examination from all ewes in Groups 1, 2, and 3. Precolostral blood samples were obtained from all lambs tiediately after parturition or following surgical recovery of the fetus. All blood samples collected were examined for the presence of PI-3 SN antibody by the microtiter technique (8).
JULY-AUGUST
1974 VOL. 2 NO. 1.2
THERIOGENOLOGY
Collection of lamb samples. Lambs from Groups 2 and 3 were recovered after parturition. Lambs from Group 1 were surgically recovered. Portions of lung, heart, spleen, liver, kidney, thymus, adrenal gland, prescapular lymph node and the abomasal content were collected from lambs in Groups 1 and 2. The fetal placenta was collected from lambs in Group 2 and placentomes were so-gically collected from ewes in Group 1. At the time of surgical removal of the fetus, abomasal contents were cultured on sheep blood agal plates for the presence of pathogenic bacteria. The placenta, cotyledons, and placentomes were ground in sterile phosphate buffered saline and individually cultured on sheep blood agar plates. All other tissues were combined and ground in sterile phosphate buffered saline and cultured on sheep blood agar plates. All the remaining tissue suspensions were frozen and preserved for virus isolation. Portions of these same tissues were fixed in 10% phosphate buffered formalin solution for histopathologic examination. Virus isolation. Virus isolation procedures were used on the tissues collected from the lambs in Groups 1 and 2 and from 1 lamb in Group 3. The tissues were ground in sterile phosphate buffered saline and .2 ml of the homogenate and .2 ml of a 1:lO dilution of the homogenate were passed 3 times in BEK cell cultures. Identification of the virus recovered was made by typical CPE and hemadsorption of bovine erythrocytes.
RESULTS Clinical observation. The ewes in Group 1 did not show visible signs of illness following intratracheal inoculation. Rectal temperatures in Groups 1 and 3 remained in a normal range after virus inoculation. A slight leukopenia was obsenred in ewes in Group 1 (Figure 1). The leukopenia began 2 days after virus inoculation and lasted throughout the observation period. The differential WBC counts revealed a moderate lymphocytosis in the ewes in Group 1. Differential and total WBC counts of ewes in Group 3 remained constant (Figure 1). Ewe 435 in Group 3 delivered 2 full term live lambs, and with assistance, ewe 396 in Group 3 delivered 1 dead lamb. Ewes 318 and 288 in Group 1 did not deliver lambs. Two full term lambs were surgically removed from both ewes 414 and 280 in Group 1. The lambs were alive at the time of surgery and were killed immediately after delivery and tissues and blood were collected for histopathologic, microbiologic, and serologic examination. Ewe 336 in Group 2 delivered 2 lambs, 1 of which was born alive and died immediately after parturition and the second was born dead and autolysis indicated the lamb had died approximately 36 hours prior to birth. Serologic examination. All ewes in Group 1 developed a rise in PI-3 SN antibody titer following inoculation (Table 1). Ewe 336 in Group 2 developed at least a sevenfold antibody rise 43 days after intrauterine fetal inoculation. Ewes in Group 3 did not show a PI-3 SN antibody titer rise. All lambs from Groups 1 and 3 had negative PI-3 SN precolostral antibody titers at the 1:4 dilution (Table 1). In Group 2 lamb 336A
JULY-AUGUST 1974
VOL. 2 NO. l-2
31
THERIOGENOLOGY had developed a 1:2048 SN antibody titer and 336B had developed a 1:512 SN antibody titer (Table 1). Microbiologic examination. Virus was not recwered from fetal tissue in Groups 1 and 3. Virus was not recovered from lamb 336A in Group 2, but was isolated from the tissues of lamb 336B. No known pathogenic bacteria were isolated from the fetal fluid and tissues collected. Pathologic examination. There were no gross or microscopic lesions in the lambs recovered from ewes in Group 1. The microscopic lesions in the two fetuses of Group 2 were similar to the lesions described previously in bovine fetuses (4, 7). There was a chronic interstitial pneumonia interspersed between areas of normal lung in both fetuses. Lymphoreticular nodules were evident in the lungs. Lamb 396 which died following forced extraction from the ewe had a congenital diaphragmatic hernia, with the abdominal viscera obliterating the thoracic cavity.
DISCUSSION Earlier studies with cattle (4, 7) have indicated that intrauterine inoculations of the bovine fetus with PI-3 virus can produce fetal disease and death. Evidence also indicates that pregnant immune heifers inoculated parenterally resist transplacental transmission of PI-3 virus (7); In this study ewes that received intratracheal inoculations failed to develop a fetal infection. All of the sheep used had preexisting PI-3 SN antibody titers, therefore it may not be possible to infect the fetus by way of the immune ewe. Circulating PI-3 antibody may neutralize the virus when it is introduced in a parenteral route of inoculation. Data on hematological values in sheep have been reported (9) and the leukocyte responses in this study were compared to the mean values reported. A leukocytosis or a leukopenia may develop in a PI-3 virus infection (10) and in this study a leukopenia was observed (Figure 1). A lymphocytosis has been reported in PI-3 virus infections in bovines (111, and a moderate lymphocytosis developed in the ewes in Group 1. None of the ewes in Group 1 showed signs of illness after inoculation and body temperature remained in normal range. Ewes 318 and 288 in Group 1 did not deliver lambs, but were probably not pregnant at the time of inoculation. Ewe 336 was not directly inoculated with the virus, but arter intrauterine fetal inoculation the PI-3 SN antibody titer revealed at least a sevenfold rise 43 days after the fetal inoculations. It is possible the uterus was contaminated wit1 virus at the time of inoculation. It is also possible that the vin s may cross the placenta from the fetus to the ewe and enter directly inta maternal circulation where it stimulates an anamnestic response. Fetal immune competency has been established in bovine fetuses with PI-3 virus (4) and in this study fetal immune competency to PI-3 virus was observed in the ovine fetuses inoculated in utero. Neither
32
JULY-AUGUST 1974
VOL. 2 NO. 1.2
THERIOGENOLOGY of the 2 fetuses inoculated-in utero were aborted, but 336A was dead at the time of parturitionand autolysis indicatedthe lamb had been dead 36 hours. Lamb 336B died at the time of parturition. Both fetuses were infectedwith PI-3 virus as indicatedby the immunologicalresponse, the recovery of PI-3 virus from lamb 336B, and the microscopiclung lesions. ACKNOWLPDGKMKNTS Publishedwith-the approval of Director,Wyoming Agricultural RxperimentStation,.asJournal Article No. JA 627. Conducted in cooperationtith Western Regional Research project W-112, ReproductivePerformancein Cattle and Sheep. The authors thank Mrs. Linda Raymond and Mrs. Inez Johnson for technicalassistancein serologicand histopathologicprocedures,and Dr. Newton Kingston for aid in translationof foreign literature.
ThenmnbersofanimaLsYqcxtedintk3pqerarerelativle1ysmall. Thepositim fitdingthattiavinefetusis~lqi~yamptent
toreqxdtotheparainfluenza3virusonthe66thdayofgestation isjustifi~onthebasisoft&e zFespmeOf~l~.~~ mightshavthtsanE?laIlb3kulldIlot~toiMculationofthrevirus. Hmwer,thefactrenains thatsanelambsdoresponl~imnxneonpetenceisestablish&.
ofthissama&or'sexperienceincattle(seeEdeference 7)andthat inthf?caseofmstviraldiseasestbs preEmceofcirailating~ onlyrarelyptmitsthexizu.5toreachtheplacenb.wemstassmein thisq@mnkthattheewesdidbecmeinfectsdbecauseofthe8-fold to64-foldriseintiter.
JULY-AUGUST 1974 VOL. 2 NO. 1.2
33
Daysof Gestatim
16
396
16
16
16
-
512 (4096 228
-
16
32
3096
2048 512 128
128
c4
<4 435B 39m
<4
512 336B
435A
22048
c4 <4
<4 <4
336A
418A 4148 Nolmb NolaIIb 280A 280B
LaMNo.
condition oflzmd~ atbixth
Li;l""""' Cesarean)
i%Jematural delivery) Live(Natural ckelivesy) Negative Died@ssisted delivery1
-
Negative Dead(Natu~al delivr?sy) Emitiw Live(Natuml delivety)
Ne$iti a
Negative LiveKesarean~
Precolo&al Virus senm recwexy
IkKlbS
--
_._.....
-
_ . -- . . - ..
_ .~
*Titersam expressedas the reciprocalof dilution oE serm that ne~Imlized 1000 %cID~~/~~ of PI-~ V~~US
32
435
32
32
Grcup2,inuteroinoallaticln -336 66 32
Grq3,cml~ls
512 1024 256
86 61 81
318 288 280
16 32 16 16
78
414
128
Titer of mxkralizing antikcdy," Dayspixthmlation 51 14 30
cscUp1,intratrcschedlinccula~m
me**
-c=,
Table1.~of~ewesandfetusestoinrxulationwithparainfluenza3virus.
. ..
1
g r,
8
8
z
El
c3
THERIOGENOLOGY
\
Total
JULY-AUGUST
/
I I
WBCs
1974
( x 103/mm3)
VOL. 2 NO. 1-2
35
FAILURE TO DEMONSTRATE TRANSPLACENTAL TRANSMISSION OF PARAINFLUENEA-3 VIRUS BY INOCULATION OF PREGNANT IMMUNE SHEEP Robert B. Grieve and Brinton L. Swift Division of Microbiology and Veterinary Medicine University of Wyoming, Laramie, Wyoming 82071 Reoeived for plblication Arqust 8, 1974 ABSTRACT Seven ewes with circulating PI-3 serum neutralizing (SN) antibody were divided into 3 groups. Ewes in Group 1 which consisted of 4 ewes, were inoculated intratracheslly, at midgestation with parainfluenza-3 (PI-3) virus isolated from an aborted bovine fetus. Four fetal lambs were removed surgically from 2 ewes in Group 1. These lambs were negative for precolostral PI-3 SN antibody and virus was not recovered from the tissues of the lambs. Two ewes in Group 1 did not deliver lambs. All ewes in Group 1 developed an increase in PI-3 SN antibody titers following inoculation. Two fetuses of the ewe in Group 2 were inoculated in utero-with PI-3 virus. Both lambs were stillborn and had elevated _precolostral PI-3 SN antibody titers. Virus was recovered from the tissues of 1 lamb. Group 3 consisted of 2 uninoculated ewes. All lambs born in Group 3 had negative precolostral PI-3 SN antibody titers and virus was not isolated from the fetal tissues. Pregnant immune sheep inoculated with virulent PI-3 virus resist transplacental transmission of virus. INTRODUCTION Parainfluenza-3 (PI-3) virus infections in sheep are common (1). The virus has been isolated from aborted bovine and ovine fetuses (2,3, 4), but the role of the PI-3 virus in fetal disease and abortion in ewes is unknown. Fetal disease and abortion have been demonstrated by intrauterine inoculations of virus in cattle and sheep (2,4,5). Pregnant immune heifers were found to resist transplacental transmission of PI-3 virus by parenteral inoculation with PI-3 virus recovered from an aborted bovine fetus (7). Experimental abortion has been reported in sheep by intravenous inoculation with PI-3 virus (2). This experiment was designed to study the possibility of transplacental transmission of PI-3 virus in pregnant immune ewes by intratracheal inoculation of the virus. PI-3 seronegative sheep were not available for this study. MATERIALS AND METRODS Experimental sheep. Seven aged, well nourished, western whitefaced ewes B the University of Wyoming Livestock Experimental Farm at Laramie, Wyoming. All ewes had existing PI-3 serum neutralizing (SN) antibody titers before experimental inoculation (Table 1). Rams fitted with marking harnesses were joined with the ewes for 54 days and colors on the harnesses were changed every 14 days. The ewes were con-
JULY-AUGUST
1974
VOL. 2 NO. 1.2
THERIOGENOLOGY sidered pregnant when they no longer were mounted and marked by the rams. The breeding dates were recorded and the ewes were moved to the University of Wyoming Veterinary Medical Research Center at Laramie after breeding. They were fed dehydrated alfalfa pellets daily with salt and heated water ad libitum. The ewes were separated into 3 groups according to their SN antibody titers, their breeding dates and route of inoculation. Experimental design. Group 1 (Table 1) consisted of 4 ewes, 3 of which had existing PI-3 SN antibody titers of 1:16 and 1 of 1:32. These ewes were inoculated intratracheally. Group 2 consisted of 1 ewe which had an existing PI-3 SN antibody titer of 1:32. A midline laparotomy was performed on this ewe for the purpose of fetal inoculation on the 66 day of gestation. This route of inoculation was used as a positive control to determine the response of the ovine fetus to PI-3 virus by direct inoculation. Group 3 consisted of two ewes with PI-3 SN antibody titers of 1:16 and 1:32. These ewes served as uninoculated controls. Virus. The virus was recovered from an aborted bovine fetus (4) and was previously characterized (6). The ewes in Group 1 were inoculated intratracheally with 10 ml of the isolate from bovine embryonic kidney cell cultures (BEK) containing lo5 TCIU50/ml of virus. The two fetuses in Group 2 were inoculated with 1 ml each of the same inoculum. Surgical procedures. A midline laparotomy was performed on the ewe.in Group 2 for an intrauterine fetal inoculation and on each ewe in Group 1 to recover the fetuses prior to the expected time of parturition. One million units of penicillin and 1.25.grams streptomycin were administered intramuscularly daily for four days after surgery. Clinical observations. Rectal termperatures of ewes in Groups 1 and 3 were determined every 3 days beginning 6 days before inoculation and continuing 17 days following inoculation and again at 23 and 30 days following inoculation. Blood was collected at the same time in a vacuum tube containing ethylenediametetraacetic acid for differential and total white blood cell (WBC) counts. Blood samples were collected 6 days before inoculation and 14, 30, and 51 days after inoculation from all ewes in Groups 1 and 3 for PI-3 serology. Blood samples were collected from the ewe..inGroup 2 one day before inoculation and 6, 37, and 69 days after inoculation. The ewes were observed daily throughout the gestation period. Clinical pathology. Total white blood cell (WBC) counts and differential WBC counts were determined from blood samples collected from ewes in Groups 1 and 3. A hemacytometer was used to determine total WBC counts and blood smears were stained with Giemsa stain to determine the differential WBC counts. Serological examination. Blood samples were collected for serological examination from all ewes in Groups 1, 2, and 3. Precolostral blood samples were obtained from all lambs tiediately after parturition or following surgical recovery of the fetus. All blood samples collected were examined for the presence of PI-3 SN antibody by the microtiter technique (8).
JULY-AUGUST
1974 VOL. 2 NO. 1.2
THERIOGENOLOGY
Collection of lamb samples. Lambs from Groups 2 and 3 were recovered after parturition. Lambs from Group 1 were surgically recovered. Portions of lung, heart, spleen, liver, kidney, thymus, adrenal gland, prescapular lymph node and the abomasal content were collected from lambs in Groups 1 and 2. The fetal placenta was collected from lambs in Group 2 and placentomes were so-gically collected from ewes in Group 1. At the time of surgical removal of the fetus, abomasal contents were cultured on sheep blood agal plates for the presence of pathogenic bacteria. The placenta, cotyledons, and placentomes were ground in sterile phosphate buffered saline and individually cultured on sheep blood agar plates. All other tissues were combined and ground in sterile phosphate buffered saline and cultured on sheep blood agar plates. All the remaining tissue suspensions were frozen and preserved for virus isolation. Portions of these same tissues were fixed in 10% phosphate buffered formalin solution for histopathologic examination. Virus isolation. Virus isolation procedures were used on the tissues collected from the lambs in Groups 1 and 2 and from 1 lamb in Group 3. The tissues were ground in sterile phosphate buffered saline and .2 ml of the homogenate and .2 ml of a 1:lO dilution of the homogenate were passed 3 times in BEK cell cultures. Identification of the virus recovered was made by typical CPE and hemadsorption of bovine erythrocytes.
RESULTS Clinical observation. The ewes in Group 1 did not show visible signs of illness following intratracheal inoculation. Rectal temperatures in Groups 1 and 3 remained in a normal range after virus inoculation. A slight leukopenia was obsenred in ewes in Group 1 (Figure 1). The leukopenia began 2 days after virus inoculation and lasted throughout the observation period. The differential WBC counts revealed a moderate lymphocytosis in the ewes in Group 1. Differential and total WBC counts of ewes in Group 3 remained constant (Figure 1). Ewe 435 in Group 3 delivered 2 full term live lambs, and with assistance, ewe 396 in Group 3 delivered 1 dead lamb. Ewes 318 and 288 in Group 1 did not deliver lambs. Two full term lambs were surgically removed from both ewes 414 and 280 in Group 1. The lambs were alive at the time of surgery and were killed immediately after delivery and tissues and blood were collected for histopathologic, microbiologic, and serologic examination. Ewe 336 in Group 2 delivered 2 lambs, 1 of which was born alive and died immediately after parturition and the second was born dead and autolysis indicated the lamb had died approximately 36 hours prior to birth. Serologic examination. All ewes in Group 1 developed a rise in PI-3 SN antibody titer following inoculation (Table 1). Ewe 336 in Group 2 developed at least a sevenfold antibody rise 43 days after intrauterine fetal inoculation. Ewes in Group 3 did not show a PI-3 SN antibody titer rise. All lambs from Groups 1 and 3 had negative PI-3 SN precolostral antibody titers at the 1:4 dilution (Table 1). In Group 2 lamb 336A
JULY-AUGUST 1974
VOL. 2 NO. l-2
31
THERIOGENOLOGY had developed a 1:2048 SN antibody titer and 336B had developed a 1:512 SN antibody titer (Table 1). Microbiologic examination. Virus was not recwered from fetal tissue in Groups 1 and 3. Virus was not recovered from lamb 336A in Group 2, but was isolated from the tissues of lamb 336B. No known pathogenic bacteria were isolated from the fetal fluid and tissues collected. Pathologic examination. There were no gross or microscopic lesions in the lambs recovered from ewes in Group 1. The microscopic lesions in the two fetuses of Group 2 were similar to the lesions described previously in bovine fetuses (4, 7). There was a chronic interstitial pneumonia interspersed between areas of normal lung in both fetuses. Lymphoreticular nodules were evident in the lungs. Lamb 396 which died following forced extraction from the ewe had a congenital diaphragmatic hernia, with the abdominal viscera obliterating the thoracic cavity.
DISCUSSION Earlier studies with cattle (4, 7) have indicated that intrauterine inoculations of the bovine fetus with PI-3 virus can produce fetal disease and death. Evidence also indicates that pregnant immune heifers inoculated parenterally resist transplacental transmission of PI-3 virus (7); In this study ewes that received intratracheal inoculations failed to develop a fetal infection. All of the sheep used had preexisting PI-3 SN antibody titers, therefore it may not be possible to infect the fetus by way of the immune ewe. Circulating PI-3 antibody may neutralize the virus when it is introduced in a parenteral route of inoculation. Data on hematological values in sheep have been reported (9) and the leukocyte responses in this study were compared to the mean values reported. A leukocytosis or a leukopenia may develop in a PI-3 virus infection (10) and in this study a leukopenia was observed (Figure 1). A lymphocytosis has been reported in PI-3 virus infections in bovines (111, and a moderate lymphocytosis developed in the ewes in Group 1. None of the ewes in Group 1 showed signs of illness after inoculation and body temperature remained in normal range. Ewes 318 and 288 in Group 1 did not deliver lambs, but were probably not pregnant at the time of inoculation. Ewe 336 was not directly inoculated with the virus, but arter intrauterine fetal inoculation the PI-3 SN antibody titer revealed at least a sevenfold rise 43 days after the fetal inoculations. It is possible the uterus was contaminated wit1 virus at the time of inoculation. It is also possible that the vin s may cross the placenta from the fetus to the ewe and enter directly inta maternal circulation where it stimulates an anamnestic response. Fetal immune competency has been established in bovine fetuses with PI-3 virus (4) and in this study fetal immune competency to PI-3 virus was observed in the ovine fetuses inoculated in utero. Neither
32
JULY-AUGUST 1974
VOL. 2 NO. 1.2
THERIOGENOLOGY of the 2 fetuses inoculated-in utero were aborted, but 336A was dead at the time of parturitionand autolysis indicatedthe lamb had been dead 36 hours. Lamb 336B died at the time of parturition. Both fetuses were infectedwith PI-3 virus as indicatedby the immunologicalresponse, the recovery of PI-3 virus from lamb 336B, and the microscopiclung lesions. ACKNOWLPDGKMKNTS Publishedwith-the approval of Director,Wyoming Agricultural RxperimentStation,.asJournal Article No. JA 627. Conducted in cooperationtith Western Regional Research project W-112, ReproductivePerformancein Cattle and Sheep. The authors thank Mrs. Linda Raymond and Mrs. Inez Johnson for technicalassistancein serologicand histopathologicprocedures,and Dr. Newton Kingston for aid in translationof foreign literature.
ThenmnbersofanimaLsYqcxtedintk3pqerarerelativle1ysmall. Thepositim fitdingthattiavinefetusis~lqi~yamptent
toreqxdtotheparainfluenza3virusonthe66thdayofgestation isjustifi~onthebasisoft&e zFespmeOf~l~.~~ mightshavthtsanE?laIlb3kulldIlot~toiMculationofthrevirus. Hmwer,thefactrenains thatsanelambsdoresponl~imnxneonpetenceisestablish&.
ofthissama&or'sexperienceincattle(seeEdeference 7)andthat inthf?caseofmstviraldiseasestbs preEmceofcirailating~ onlyrarelyptmitsthexizu.5toreachtheplacenb.wemstassmein thisq@mnkthattheewesdidbecmeinfectsdbecauseofthe8-fold to64-foldriseintiter.
JULY-AUGUST 1974 VOL. 2 NO. 1.2
33
Daysof Gestatim
16
396
16
16
16
-
512 (4096 228
-
16
32
3096
2048 512 128
128
c4
<4 435B 39m
<4
512 336B
435A
22048
c4 <4
<4 <4
336A
418A 4148 Nolmb NolaIIb 280A 280B
LaMNo.
condition oflzmd~ atbixth
Li;l""""' Cesarean)
i%Jematural delivery) Live(Natural ckelivesy) Negative Died@ssisted delivery1
-
Negative Dead(Natu~al delivr?sy) Emitiw Live(Natuml delivety)
Ne$iti a
Negative LiveKesarean~
Precolo&al Virus senm recwexy
IkKlbS
--
_._.....
-
_ . -- . . - ..
_ .~
*Titersam expressedas the reciprocalof dilution oE serm that ne~Imlized 1000 %cID~~/~~ of PI-~ V~~US
32
435
32
32
Grcup2,inuteroinoallaticln -336 66 32
Grq3,cml~ls
512 1024 256
86 61 81
318 288 280
16 32 16 16
78
414
128
Titer of mxkralizing antikcdy," Dayspixthmlation 51 14 30
cscUp1,intratrcschedlinccula~m
me**
-c=,
Table1.~of~ewesandfetusestoinrxulationwithparainfluenza3virus.
. ..
1
g r,
8
8
z
El
c3
THERIOGENOLOGY
\
Total
JULY-AUGUST
/
I I
WBCs
1974
( x 103/mm3)
VOL. 2 NO. 1-2
35