Vol. 25, No. 1
INFECTION AND IMMUNITY, July 1979, p. 93-97 0019-9567/79/07-0093/05$02.00/0
Serological Response of the Bovine Fetus to Bovine Viral Diarrhea Virus TALMAGE T. BROWN, JR.,t* RONALD D. SCHULTZ,4 J. ROBERT DUNCAN,§ AND STEPHEN I.
BISTNERII
Department of Microbiology and Large Animal Medicine, Obstetrics and Surgery, New York State College of Veterinary Medicine, Cornell University, Ithaca, New York 14853 Received for publication 5 February 1979
Susceptible pregnant heifers were inoculated with bovine viral diarrhea virus at 150 days of gestation and earlier. Fetuses were surgically collected at selected times after inoculation. Serum immunoglobulins were quantitated, and the presence of specific antibodies was determined. In fetuses from heifers inoculated at 150 days, immunoglobulin M (IgM) appeared approximately 2 weeks after inoculation and was followed in 7 days by IgGj. Later IgG2 was detected in the sera of three fetuses. Serum-neutralizing and complement-fixing antibodies were first detected in a fetus taken at 206 days of gestation. Fetuses taken at later times also had specific serum antibodies. Possible explanations for the appearance of serum immunoglobulin substantially before specific bovine viral diarrhea antibodies include the viral alteration of host tissues rendering them antigenic, viral activation of polyclonal B cells, and viral modulation of virus-specific lymphocytes causing specific interference with the appearance of antiviral antibodies. In one of the fetuses having IgG2, the serum also contained IgA. Placental leakage of material immunoglobulins was thought to be responsible for the presence of IgA and IgG2 in this fetus. Small quantities of IgM were found in the serum of two fetuses taken from heifers inoculated between 65 and 95 days of gestation, but specific antibodies were found in none. The bovine fetus is susceptible to infection and injury by bovine viral diarrhea (BVD) virus (4, 5, 7, 11, 25). Fetuses infected with BVD virus during the first 3 months of the 9-month gestational period more commonly die and are expelled, whereas those infected between 3 and 6 months of gestation usually survive but often have congenital malformations of the brain and eyes at birth. BVD infection after the sixth gestational month usually does not result in fetal malformations (6, 7, 11, 20). BVD serum-neutralizing (SN) antibodies in fetuses surviving infection are first detected at about 200 days of gestation (3, 7). However, immunoglobulin G (IgG) lacking neutralizing activity has been reported at 180 days of gestation in the BVD-infected fetus, suggesting immuno-
logical responsiveness to .the virus before the period of specific antibody production (3). The present report concerns a more detailed study of the serological response of the bovine fetus to BVD virus after infection of susceptible pregnant heifers between 65 and 150 days of gestation. MATERIALS AND METHODS Experimental animals and procedures. Thirtytwo pregnant Holstein-Friesian heifers with known breeding dates were housed in an isolated barn. SN
antibodies against BVD virus were absent in the sera of 30 heifers and present in the sera of two heifers used as controls. The heifers were serologically surveyed each month to insure that they remained susceptible to BVD virus. Before inoculation, the animals were moved into environmentally controlled individual isolation units. Twenty-five heifers were inoculated intravenously with 250,000 median tissue culture infective doses of the Holmes strain of BVD virus at 149, 150, or 151 days of gestation. The other seven heifers were inoculated similarly with the same amount of the virus at 65, 75, 80, 85, 95, or 115 days of gestation. For 14 days after inoculation, rectal temperatures were recorded every 8 h, and blood was collected daily for leukocyte counts and for determining SN antibody titers. After this initial period, serum samples were
t Present address: National Animal Disease Center, Science and Education Administration, Agricultural Research, U.S. Department of Agriculture, Ames, IA 50010. t Present address: Department of Microbiology, College of Veterinary Medicine, Auburn University, Auburn, AL 36830. § Present address: Agriculture Canada, Animal Diseases Research Institute, Ottawa, Ontario K2H8P9, Canada. 11 Present address: Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Minnesota, St. Paul, MN 55108.
93
94
INFECT. IMMUN.
BROWN ET AL.
collected weekly until the heifers were removed from the experiment. At sequential times after inoculation (Table 1), fetuses were surgically removed by cesarean section from their dams. They were exsanguinated immediately, and the serum was stored at -70OC for the various assays. Two of the susceptible heifers were allowed to calve normally, and serum was collected from the calves before they ingested colostrum. Virus. The cytopathogenic Holmes strain of BVD virus was used in the experiment. The source and preparation of the virus has been described previously (4, 21). Cells. Fetal bovine bone marrow cells provided by J. W. Kendrick (School of Veterinary Medicine, University of California, Davis) were grown in Eagle min-
imal essential medium (GIBCO Laboratories, Grand Island, N.Y.) supplemented with 0.05% lactalbumin hydrolysate, 100 U of penicillin per ml, 100 ,tg of streptomycin per ml, 25 U of mycostatin per ml, and 10% fetal calf serum (GIBCO Diagnostics, Madison, Wis.). They were maintained in medium 199 with Hanks balanced salt solution (GIBCO Diagnostics) supplemented with 0.5% lactalbumin hydrolysate, the same antibiotic combination as in the growth media, and 2% lamb's serum (GIBCO Diagnostics). Primary calf testicular cells were grown in minimal essential medium supplemented with 50 ,ug of gentamicin per ml, 12.5 Ag of amphotericin B (Fungizone) per ml, and 10) fetal calf serum. The same media with 2% fetal calf serum was used for maintenance.
Immunoglobulin quantification. Immunoglobu-
TABLE 1. Serum immunoglobulin and specific antibody production in bovine fetuses after infection with BVD virus Gestational age (days) at:
SN' anti-
CF' an-
IgM
IgG,
IgG2
4 7 12 13 14
0 0 0.04 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
163 165 166 167 167
14 15 16 17 17
0.1 0.07 0.23 0.60 0.22
0 0 0 0.22
0 0 0 0
0 ND 0 0 0
0 ND 0 ND 0
171 174 177 178 191
21 24 27 28 42
0.35 0.20 0.77 0 0.20
0 0.29 0.06 0 0.30
0 0 0.027 0 0
0 0 0 0 0
ND 0 0
57
150 150 150 150 149
0 0
ND ND ND 0 0
1901 53 50 45
150 149 149 150
206 219 232 290
56 70 83 140
0.55
INFECTION AND IMMUNITY, July 1979, p. 93-97 0019-9567/79/07-0093/05$02.00/0
Serological Response of the Bovine Fetus to Bovine Viral Diarrhea Virus TALMAGE T. BROWN, JR.,t* RONALD D. SCHULTZ,4 J. ROBERT DUNCAN,§ AND STEPHEN I.
BISTNERII
Department of Microbiology and Large Animal Medicine, Obstetrics and Surgery, New York State College of Veterinary Medicine, Cornell University, Ithaca, New York 14853 Received for publication 5 February 1979
Susceptible pregnant heifers were inoculated with bovine viral diarrhea virus at 150 days of gestation and earlier. Fetuses were surgically collected at selected times after inoculation. Serum immunoglobulins were quantitated, and the presence of specific antibodies was determined. In fetuses from heifers inoculated at 150 days, immunoglobulin M (IgM) appeared approximately 2 weeks after inoculation and was followed in 7 days by IgGj. Later IgG2 was detected in the sera of three fetuses. Serum-neutralizing and complement-fixing antibodies were first detected in a fetus taken at 206 days of gestation. Fetuses taken at later times also had specific serum antibodies. Possible explanations for the appearance of serum immunoglobulin substantially before specific bovine viral diarrhea antibodies include the viral alteration of host tissues rendering them antigenic, viral activation of polyclonal B cells, and viral modulation of virus-specific lymphocytes causing specific interference with the appearance of antiviral antibodies. In one of the fetuses having IgG2, the serum also contained IgA. Placental leakage of material immunoglobulins was thought to be responsible for the presence of IgA and IgG2 in this fetus. Small quantities of IgM were found in the serum of two fetuses taken from heifers inoculated between 65 and 95 days of gestation, but specific antibodies were found in none. The bovine fetus is susceptible to infection and injury by bovine viral diarrhea (BVD) virus (4, 5, 7, 11, 25). Fetuses infected with BVD virus during the first 3 months of the 9-month gestational period more commonly die and are expelled, whereas those infected between 3 and 6 months of gestation usually survive but often have congenital malformations of the brain and eyes at birth. BVD infection after the sixth gestational month usually does not result in fetal malformations (6, 7, 11, 20). BVD serum-neutralizing (SN) antibodies in fetuses surviving infection are first detected at about 200 days of gestation (3, 7). However, immunoglobulin G (IgG) lacking neutralizing activity has been reported at 180 days of gestation in the BVD-infected fetus, suggesting immuno-
logical responsiveness to .the virus before the period of specific antibody production (3). The present report concerns a more detailed study of the serological response of the bovine fetus to BVD virus after infection of susceptible pregnant heifers between 65 and 150 days of gestation. MATERIALS AND METHODS Experimental animals and procedures. Thirtytwo pregnant Holstein-Friesian heifers with known breeding dates were housed in an isolated barn. SN
antibodies against BVD virus were absent in the sera of 30 heifers and present in the sera of two heifers used as controls. The heifers were serologically surveyed each month to insure that they remained susceptible to BVD virus. Before inoculation, the animals were moved into environmentally controlled individual isolation units. Twenty-five heifers were inoculated intravenously with 250,000 median tissue culture infective doses of the Holmes strain of BVD virus at 149, 150, or 151 days of gestation. The other seven heifers were inoculated similarly with the same amount of the virus at 65, 75, 80, 85, 95, or 115 days of gestation. For 14 days after inoculation, rectal temperatures were recorded every 8 h, and blood was collected daily for leukocyte counts and for determining SN antibody titers. After this initial period, serum samples were
t Present address: National Animal Disease Center, Science and Education Administration, Agricultural Research, U.S. Department of Agriculture, Ames, IA 50010. t Present address: Department of Microbiology, College of Veterinary Medicine, Auburn University, Auburn, AL 36830. § Present address: Agriculture Canada, Animal Diseases Research Institute, Ottawa, Ontario K2H8P9, Canada. 11 Present address: Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Minnesota, St. Paul, MN 55108.
93
94
INFECT. IMMUN.
BROWN ET AL.
collected weekly until the heifers were removed from the experiment. At sequential times after inoculation (Table 1), fetuses were surgically removed by cesarean section from their dams. They were exsanguinated immediately, and the serum was stored at -70OC for the various assays. Two of the susceptible heifers were allowed to calve normally, and serum was collected from the calves before they ingested colostrum. Virus. The cytopathogenic Holmes strain of BVD virus was used in the experiment. The source and preparation of the virus has been described previously (4, 21). Cells. Fetal bovine bone marrow cells provided by J. W. Kendrick (School of Veterinary Medicine, University of California, Davis) were grown in Eagle min-
imal essential medium (GIBCO Laboratories, Grand Island, N.Y.) supplemented with 0.05% lactalbumin hydrolysate, 100 U of penicillin per ml, 100 ,tg of streptomycin per ml, 25 U of mycostatin per ml, and 10% fetal calf serum (GIBCO Diagnostics, Madison, Wis.). They were maintained in medium 199 with Hanks balanced salt solution (GIBCO Diagnostics) supplemented with 0.5% lactalbumin hydrolysate, the same antibiotic combination as in the growth media, and 2% lamb's serum (GIBCO Diagnostics). Primary calf testicular cells were grown in minimal essential medium supplemented with 50 ,ug of gentamicin per ml, 12.5 Ag of amphotericin B (Fungizone) per ml, and 10) fetal calf serum. The same media with 2% fetal calf serum was used for maintenance.
Immunoglobulin quantification. Immunoglobu-
TABLE 1. Serum immunoglobulin and specific antibody production in bovine fetuses after infection with BVD virus Gestational age (days) at:
SN' anti-
CF' an-
IgM
IgG,
IgG2
4 7 12 13 14
0 0 0.04 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
163 165 166 167 167
14 15 16 17 17
0.1 0.07 0.23 0.60 0.22
0 0 0 0.22
0 0 0 0
0 ND 0 0 0
0 ND 0 ND 0
171 174 177 178 191
21 24 27 28 42
0.35 0.20 0.77 0 0.20
0 0.29 0.06 0 0.30
0 0 0.027 0 0
0 0 0 0 0
ND 0 0
57
150 150 150 150 149
0 0
ND ND ND 0 0
1901 53 50 45
150 149 149 150
206 219 232 290
56 70 83 140
0.55
