Dec 7, 1976 - titers and Ig classes of Pr and TGE virus-neutralizing antibodies in ... varying but lesser amounts of antibody associated with the IgG class. Such.
Vol. 16, No. 3 Printed in U.S.A.
INFECTION AND IMMUNTY, June 1977, p. 961-966 Copyright © 1977 American Society for Microbiology
Immunoglobulin Classes of Antibodies in Milk of Swine After Intranasal Exposure to Pseudorabies Virus or Transmissible Gastroenteritis Virus1 LINDA J. SAIF* AND EDWARD H. BOHL Department ofVeterinary Science, Ohio Agricultural Research and Development Center, Wooster, Ohio 44691 Received for publication 7 December 1976
Experiments were conducted to evaluate whether infection of the respiratory tract of pregnant swine with pseudorabies (Pr) virus would induce the secretion of immunoglobulin A (IgA) antibodies in their milk as was observed after enteric infection with transmissible gastroenteritis (TGE) virus. The immune response of sows to Pr virus inoculated intranasally and to TGE virus inoculated orally/ intranasally or via a natural infection was studied. Emphasis was placed upon titers and Ig classes of Pr and TGE virus-neutralizing antibodies in colostrum and milk. All animals exposed to Pr virus (alone or in combination with TGE virus) developed Pr-neutralizing antibody titers in both serum and milk. Pr antibody titers were generally higher in colostrum than in serum, but the opposite was true in milk compared with serum, with milk titers declining markedly during lactation. In contrast, TGE antibody titers in milk from experimentally or naturally infected sows usually remained higher than the corresponding serum titers and persisted at relatively constant levels throughout lactation. Gel filtration studies of milk indicated that the antibody activity against Pr virus was associated almost entirely with IgG fractions, with small amounts of antibody detectable in IgM fractions in colostrum from two of nine sows. By comparison, TGE antibodies were primarily of the IgA class, with varying but lesser amounts of antibody associated with the IgG class. Such results suggest that viral infection of the intestinal tract of the sow, but not the upper respiratory tract, stimulates the secretion ofIgA antibodies in the milk.
In previous investigations, transmissible gastroenteritis (TGE) virus has been used as a model to study the immune response of swine to an enteric viral infection. The virus is highly suited to this type of study since it infects primarily the epithelial cells of the small intestine, producing villous atrophy and a malabsorption syndrome (6). Results of these studies have indicated that TGE antibodies of the immunoglobulin A (IgA) class are produced in the milk only after an infection of the intestinal tract of the sow, as occurred after natural infection or oral administration of TGE virus. In contrast, parenteral injection of virus (intramuscularly or intramammarily) resulted in predominately IgG TGE antibodies in the milk (6-8, 23). Recent reports have confirmed such findings in other species, and oral administration of various antigens has resulted in the appearance of IgA antibodies in the milk, often ' Approved for publication as journal article no. 195-76 of the Ohio Agricultural Research and Development Center, Wooster, Ohio.
in the absence of serum antibodies (1, 10, 18, 19, 26). From these observations it has been suggested that IgA-producing cells may migrate from the gut to the mammary gland. Other studies have emphasized the importance of a common mucosal immunological system whereby cells from the bronchus-associated lymphoid tissue have the potential to repopulate the gut with IgA-containing cells (3, 22). If such a common system exists, one might expect to find IgA antibodies produced at a distant secretory site (mammary gland) after local antigenic stimulation of either the gut or respiratory tract. To explore this hypothesis, the Ig classes of antibodies produced in the milk of the sow after local stimulation of the respiratory tract were determined. Pseudorabies (Pr) virus was used as a model for a local respiratory infection since it has been shown to undergo primary multiplication in the upper respiratory tract of infected swine (11, 16). As a control, some sows were also infected with TGE virus, and the Ig classes of both Pr and TGE antibodies in the milk were determined. 961
962
SAIF AND BOHL
INFECT. IMMUN.
MATERIALS AND METHODS Viruses. (i) TGE. The virulent strain of TGE virus (Miller no. 3 strain, M-3) used in this study was described in detail previously (6). The virus suspension was of gut origin and had a titer of about 105 50% pig-infective doses per ml. Viral neutralization tests were conducted with a high-cell-culturepassage Purdue strain (6). (ii) Pr. The Sullivan strain of Pr virus was obtained from Donald Gustafson of Purdue University. This strain was used for infecting swine and for conducting the viral neutralization test. It was propagated in primary porcine kidney (PK) cell cultures and had a titer of approximately 3 x 107 plaqueforming units per ml. Infection of experimental animals. (i) Inoculation with Pr virus. Animals experimentally infected with TGE or Pr virus were housed individually in isolation rooms. A total of nine serologically negative pregnant swine were inoculated intranasally (i.n.) with 2 to 4 ml of either a 1:1,000 (sows 305, 325, 15-12, 10-8, 4-7) or 1:10,000 (sows 25-2, 87, 4-11) dilution of Pr virus at 7 to 48 days prefarrowing. Three sows that did not show clinical signs after the first exposure were reexposed 6 to 7 days later. The precise day prefarrowing when each sow was exposed is indicated in Table 1. Virus was expelled into the nasal passageways of all nine swine with a 5inch(ca. 13-cm)-long blunt needle and syringe. Five animals (305, 325, 15-12, 10-8, and 4-7) were additionally i.n. exposed with an atomizer (DeVilbiss 152, DeVilbiss Co., Somerset, Pa.) in an attempt to introduce the virus deeper into the respiratory tract. (ii) Inoculation with TGE virus. The sows that were exposed to TGE virus and the times of exposure are summarized in Table 1. Two sows used in the study (25-2 and 4-11) were seropositive for TGE before inoculation and were considered to have been naturally infected with TGE previously. One of these sows (25-2) was experimentally (orally/i.n.) reexposed to 10 ml of the virulent M-3 virus as described previously (6). Two other sows (325 and 47) that were seronegative to TGE were exposed to M3 virus at 15 to 19 days prefarrowing: one sow (325) orally/i.n. and the other orally via a stomach tube. The remaining four sows were also seronegative for TGE and were exposed only to Pr virus. Collection and preparation of specimens. Methods for the collection and processing of serum, colostrum, and milk samples were the same as those described previously (6, 23). Antibody titrations. A plaque reduction test was used to detect TGE and Pr virus-neutralizing antibodies in various samples (6). Antibody titers were expressed as the reciprocal of the sample dilution resulting in an 80% reduction in plaques. Antisera. Preparation of rabbit antisera against porcine IgA, IgG, and IgM was done as described in an earlier report (23). Rabbit antisera to porcine IgA, IgG, and IgM were rendered monospecific for y, and ,u chains by adsorption with glutaraldehydeimmobilized heterologous Ig prepared according to the procedure of Avrameas and Ternynck (2). In some instances, light-chain cross-reactivity was rea,
moved by passing antisera through cyanogen bromide-activated Sepharose 4B (Pharmacia, Uppsala, Sweden) affinity columns coupled to the heterologous Ig. Antisera absorbed in this manner showed only a single line when tested against porcine serum or colostrum by immunoelectrophoresis or immunodiffusion. Gel filtration. Gel filtration column chromatography was performed with Sephadex G-200 and 0.1 M tris(hydroxymethyl)aminomethane-0.2 M NaCl buffer, pH 8, as described previously (23). Threemilliliter fractions were collected, and their optical density at 280 nm was determined. Portions of individual tubes were filtered through 0.45-,um-poresize membrane filters (Millipore Corp., Bedford, Mass.) and tested for TGE and Pr antibodies. Ig's present in the samples were determined by immunodiffusion against monospecific antisera as described previously (23). Immunodiffusion. The micromodification of Ouchterlony's method was used for double immunodiffusion. The 0.8% agarose gel used was prepared in 0.05 M barbital buffer, pH 8.6. RESULTS Table 1 provides information on the i.n. inoculation of eight pregnant swine with Pr and, in some cases, TGE virus and on the resulting Pr and TGE antibody titers in serum and milk. All sows had clinical signs 3 to 4 days after final exposure to Pr virus, including coughing, elevated temperature, and loss of appetite. One sow (25-4) aborted six dead fetuses at 18 days postexposure, 13 days before the expected parturition date, and another (4-7) had five mummified fetuses among her litter of four live pigs. By using crown-rump length as an estimate of fetal age, it was determined that these mummified fetuses died about 7 to 22 days after inoculation of the sow with Pr virus. At farrowing, Pr titers were generally higher in the colostrum (4 to 700) than serum (4 to 96), but by 4 to 6 days postfarrowing (DPF), titers were higher in serum than in milk in seven of eight animals (Table 1). At this time, titers in the milk had declined markedly and ranged from only 3.3 to 180. Pr antibody titers in milk and serum continued to decline throughout lactation (except sow 15-12) such that by 35 to 40 DPF milk titers were only 1.2 to 4.5. In contrast, TGE antibody titers in the milk of the naturally or experimentally infected sows generally remained higher than the analogous serum titers and remained at relatively constant
levels throughout lactation. When these samples were chromatographed on Sephadex G-200, elution profiles typical of those shown in Fig. 1 were obtained. Antibody activity against Pr virus was associated almost entirely with the IgG peak (third-peak fractions), though in colostrum from two sows (305,
VOL. 16, 1977
Ig ANTIBODY CLASSES IN SWINE MILK
963
TABLE 1. Pr and TGE antibody titers in serum and milk of swine after i.n. exposure to Pr virus alone or to Pr and TGE virus Antibody titer Sow no.
87
Virus 748 day p Sample testrrowingay prePr
TGE 25-2
Pr
TGE 4-11
Pr
TGE 305
Pr
TGE 325
Pr
TGE 15-12
Pr
TGE 10-8
4-7
Pr
Pr TGE
a b
Serum Milk Serum Milk Serum Milk
Serum Milk Serum Milk
3-6 days postfar-
rowing
rowing
96 320
310 25
86
700
400 180
320 20
96 4.5
3,800 4,600 86 370
450 2,500 100 27
320 1,150 85
450 1,024 25 1.2
