Specific antibody to Haemophilus somnus in the bovine uterus following intramuscular immunization.

Sources of anti-Haemophilus somnus antibody in bovine uterine secretions following intramuscular immunization and subsequent intrauterine inoculation of killed H. somnus were investigated. Holstein cattle (n = 21) were immunized with a 270-kDa outer membrane protein from H. somnus (omp-270) by intramuscular injection. At estrus, the cattle were given an intrauterine inoculum of a heat-killed suspension of a homologous strain of H. somnus containing omp-270 (n = 7), a heterologous strain of H. somnus lacking omp-270 (n = 7), or phosphate-buffered saline (n = 7). Uterine secretions were sampled by saline lavage immediately prior to inoculation and at 6, 24, 48, 72, 96, and 120 h after inoculation. Immunoglobulin G subclass I (IgG1) and IgG2 antibody specific for omp-270 were detectable in estrous uterine secretions of all systemically immunized cattle from which an adequate sample was obtained. IgM antibody specific for omp-270 was detected in serum following immunization but was not consistently detected in the uterine secretions of any animal. IgA antibody specific for omp-270 was not detectable in either serum or uterine secretions following immunization or intrauterine inoculation. Ratios of antibody to immunoglobulin and ratios of immunoglobulin to albumin in serum and uterine secretions indicated that about half the IgG1 and essentially all the IgG2 in secretions originated in the serum. Relative titers of IgG1 and IgG2 omp-270-specific antibodies in the uterine lumen and serum gave no evidence for selective transport of either subclass from serum into local secretions. Neither heterologous nor homologous intrauterine inocula detectably altered the serum contribution to antibody in uterine secretions within the sampling period. On the basis of these results, development of a systemic IgG2 antibody response may provide the basis for local immunological protection in the bovine reproductive tract.

Neutrophil migration into the bovine uterine lumen following intrauterine inoculation with killed Haemophilus somnus.

Polymorphonuclear neutrophils (PMN) in bovine uterine flushings following intrauterine deposition of killed bacteria were measured and the effect of immune status on the influx of PMN into the uterine lumen during oestrus was determined. Holstein heifers were immunized with a 270-kDa outer-membrane protein (omp-270) from Haemophilus somnus. During oestrus, immunized heifers (n = 21) received an intrauterine inoculum of either a heat-killed suspension of a homologous strain of H. somnus containing omp-270 (n = 7), a heterologous strain of H. somnus lacking omp-270 (n = 7), or phosphate-buffered saline (n = 7). Five additional heifers were inseminated with extended bovine semen. Uterine contents were collected in saline lavage immediately before inoculation (t0) and at 6, 24, 48, 72, 96, and 120 h after inoculation. The semen-inoculated heifers were lavaged only at t120. All groups experienced PMN infiltration which peaked 6 h after inoculation and tended to decline thereafter. Differences were not observed between treatment groups, indicating that neither bacterial inoculation nor immune status was as important in eliciting PMN effusion as the flushing procedure itself.

Characterization of heteromyeloma fusion partners which promote the outgrowth of porcine hybridomas.

Production of porcine monoclonal antibodies for use in research and immunotherapy has been hampered by the lack of suitable fusion partners which promote high efficiencies of hybridoma out-growth and immunoglobulin synthesis. To overcome these obstacles, five heteromyeloma fusion partners (HM-1,2,3,4 and 5) were constructed by successively fusing porcine lymphocytes with murine myeloma cells or murine x bovine heteromyeloma cells. Following section of hypoxanthine/aminopterin/thymidine (HAT)-sensitive mutants, karyotypes, growth rates and surface phenotypes of the heteromyelomas were determined. Karyotyping revealed an increase in the mean number of chromosomes present in HM-1,4 and 5 cells. Peak doubling times of the parental and HM cells ranged between 12.2 and 17.4 h. Uisng flow microfluorimetry and monoclonal antibodies specific for class I/II major histocompatability antigens, it was determined that the surface phenotype of HM-1,2,3,4 and 5 resembled that of the parental murine X63 myeloma cells. HM 1,2,3,4 and 5 were evaluated for their abilities to serve as fusion partners. Highest percentages of hybrid outgrowth (37%) and immunoglobulin synthesis (52%) were observed when HM-1 was fused with procine lymphocytes. When cloned, percentage of outgrowth and immunoglobulin synthesis increased if HM-1 and HM-2 were used as fusion partners. Cryopreservation of HM-1 and HM-2 did not adversely affect their abilities to promote hybrid outgrowth or immunoglobulin synthesis. During the first week following fusion of porcine lymphocytes with heteromyelomas, murine thymocytes were found to be essential for survival of the nascent hybrids. To confirm that immunoglobulin secreted by hybridomas was of porcine and not murine or bovine origin, culture supernates were subjected to SDS gel electrophoresis, electroblotted and identified. using species-specific isotyping reagents. Two of four cell lines tested secreted porcine light chains and one of four cell lines secreted whole IgM molecules. This paper is the first to describe porcine heteromyelomas for use as fusion partners. Similar to findings of human and bovine studies, our data suggest that heteromyeloma fusion partners perform better than rodent myelomas for creating hybridomas synthesizing porcine immunoglobulin.