Immunization and diagnosis in bovine reproductive tract infections.
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Abortion, Veterinary↗
Biomedical subjects
L B Corbeil
Publications and source records attributed to L B Corbeil.
Female reproductive tract immunity in bovine trichomoniasis.
PROBLEM: Mechanisms of protective immunity in the female reproductive tract are poorly understood. For sexually transmitted diseases, bovine trichomoniasis is a useful model because it resembles human trichomoniasis to some extent, and antibodies play an important role in protection against these extracellular parasites. Protective efficacy was compared in animals with genital responses of predominantly immunoglobulin G (IgG) or predominantly IgA antibodies to a purified surface antigen of Tritrichomonas foetus. METHOD OF STUDY: Immunization of mice by various routes with immunoaffinity-purified T. foetus surface antigen (TF1.17) or killed cells was used to define the best routes and antigen combinations to give predominantly IgG or IgA antibodies to TF1.17 antigens in genital secretions. Cattle were then immunized either subcutaneously (SC) two times with TF1.17 antigen and once SC with killed T. foetus or twice SC with TF1.17 antigen and once intravaginally with killed T. foetus. All immunizations were in Quil A adjuvant. Controls were not immunized. Animals were challenged intravaginally with 10(6) T. foetus 3 weeks after the third immunization. Vaginal mucus was collected weekly for culture and antibody assays. Serum was collected weekly, and uterine secretions were collected at 10 weeks post-challenge. Tissues were fixed at 10 weeks also. RESULTS: Murine studies showed systemic priming with vaginal boosting gave the highest genital IgA responses. In cattle, systemic immunization (group S) induced high IgG1 antibody levels in vaginal secretions. Systemic priming with vaginal boosting (group S/V) primed for an anamnestic vaginal IgA response after challenge with T. foetus. Cattle with predominantly IgG or predominantly IgA responses in vaginal secretions either did not become infected or cleared infection faster than controls. Uterine IgA responses at 10 weeks were highest in the vaginally boosted group, but other responses were not different from the controls at this time point. Microscopic examination of genital tissues showed subepithelial infiltration of mononuclear cells in all groups. Lymphoid aggregates or nodules were detected in vaginal sections in cattle of groups S/V and C as well as in uterine sections of all animals in all three groups. CONCLUSIONS: Both IgG and IgA antibodies to T. foetus superficial antigen were associated with protection. The timing of the response was related to the time of clearance. Lymphoid organization in the vagina and uterine tissues suggested development of mucosal inductive sites.
Animals↗
Genital and systemic immune responses in a murine model of Tritrichomonas foetus infection.
A reliable laboratory animal model would be useful for the study of immune responses to trichomoniasis, a sexually transmitted disease of human beings and cattle. Murine models are available, but pretreatment with estrogen is used, which may influence immune responses. To evaluate whether vaginal trichomoniasis could be established in nonestrogenized mice and to define the immune responses associated with the infection, CD1 and BALB/c mice were studied with or without estrogen treatment prior to inoculation with Tritrichomonas foetus. Tritrichomonas Foetus was cultured from the vagina and uterus of both estrogen-treated and untreated control mice for up to 26 wk. The infection was sustained better in BALB/c than in CD1 mice, suggesting that the former strain was most susceptible. In CD1 mice, infection was sustained less well in estrogen-treated than in untreated control mice, but there was no difference between treatment groups of BALB/c mice. IgA and IgG antibodies in vaginal secretions, uterine secretions, and serum specific for a surface antigen of T. foetus (TF1.17) were measured by enzyme-linked immunosorbent assay. In infected CD1 mice, vaginal IgA and IgG antibodies were detected by 8 wk postinoculation (PI). In infected BALB/c mice, vaginal IgA and IgG antibodies were detected by 12 wk PI. Uterine IgG responses predominated over IgA in estrogen-treated and untreated CD1 and BALB/c mice. There were high levels of IgG, but relatively no IgA in the sera of CD1 and BALB/c mice. Overall, the highest IgA response was in the vaginal secretions of infected CD1 mice, and some animals of this strain cleared the infection. These results show that a chronic trichomonad infection was established in mice without prior treatment with estrogen. The infection was associated with antibody responses in reproductive secretions and serum. This animal model will be useful in studying immunization to protect against trichomoniasis in mice not immunocompromised by estrogen.
Animals↗
Transformation of a virulence associated gene of Haemophilus somnus into a strain lacking the gene.
The role of a 76 kDa surface antigen (p76) of Haemophilus somnus in virulence was investigated. The p76 gene from a virulent isolate of H. somnus (strain 2336) was introduced into an asymptomatic carrier strain (129Pt) lacking this gene. This was accomplished by the development of a system for genetic exchange in H. somnus. The cloned p76 gene was inserted into the broad host range vector pLS88, electroporated into H. influenzae for modification and then into the H. somnus strain 129Pt. The recombinant plasmid was characterized from selected transformants and expression of the p76 protein was demonstrated by Western immunoblotting. However, transformants were not serum resistant and surface exposure of the recombinant protein could not be detected, suggesting that additional genetic elements might be required for export.
Antigens, Bacterial↗
Haemophilus somnus immunoglobulin binding proteins and surface fibrils.
The high-molecular-weight (HMW) immunoglobulin binding proteins (IgBPs) of Haemophilus somnus and a 76-kDa surface protein (p76) are found in serum-resistant virulent strains but not in several serum-sensitive strains from asymptomatic carriers. For the first time, p76 was shown to be an IgBP also. This was done by competitive inhibition studies with affinity-purified antidinitrophenol (anti-DNP) and DNP to ensure that binding was not antigen specific. The HMW IgBPs, but not the p76 IgBP, were partially purified from concentrated culture supernatant in detergent by fluid-phase liquid chromatography with a gel filtration column. Membrane extraction studies showed that p76 predominated in the Sarkosyl-soluble fraction of the bacterial cell pellet. Since integral outer membrane (OM) proteins are Sarkosyl insoluble, this is consistent with our previous finding that implicated p76 as a peripheral OM protein. The HMW IgBPs were found predominantly in the Sarkosyl-soluble fraction of the culture supernatant. This suggests that they were not integral membrane proteins and that their presence in the supernatant was not due to OM blebbing. We then showed that two IgBP-positive serum-resistant virulent strains have a surface fibrillar network but that two IgBP-negative serum-sensitive H. somnus strains from asymptomatic preputial carriers do not. Fibrils on the surfaces of IgBP+ strains bound gold-labelled bovine immunoglobulin G2 (IgG2) anti-DNP, indicating that these fibrils have IgG2 binding activity. Therefore, this study shows that H. somnus has two IgBPs, including a peripheral membrane protein and a fibrillar surface network.
2,4-Dinitrophenol↗
Bovine IgG2a antibodies to Haemophilus somnus and allotype expression.
Bovine IgG2a has been implicated in protection against pyogenic infections, including those caused by Haemophilus somnus. To further investigate the role of IgG2a in defense against H. somnus, IgG1 and IgG2a antibodies were purified from antiserum against an immunodominant 40 kDa outer membrane protein (p40) of H. somnus, which was previously shown to passively protect calves against H. somnus pneumonia. The passive protective capacity of anti-p40 IgG1 or IgG2a was evaluated in vivo in calves. Purified anti-p40 IgG1 or IgG2a was incubated with H. somnus for 15 min before intrabronchial inoculation of calves. Bacteria incubated with anti-p40 IgG1 or IgG2a were inoculated into one caudal lung lobe and bacteria incubated with IgG1 or IgG2a from the respective preimmunization serum were inoculated into the contralateral lobe. The volumes of pneumonia in the right and left lungs were determined 24 h later. The difference in volume of pneumonia with H. somnus preincubated in IgG1 pre- and postimmunization anti p40 was less (16 cm3, P = 0.298) than the difference in volume of pneumonia with H. somnus preincubated in IgG2a pre- and postimmunization anti p40 (30 cm3, P = 0.146). Although the differences in lesion size between pre- and postimmunization serum were not statistically significant, the trend suggests IgG2a may be more protective than IgG1. To examine this further, the peptide specificity of these IgG1 and IgG2a antibodies to p40 was examined. After limited proteolysis of p40, IgG2a antibodies reacted with 2 peptides not recognized by IgG1 antibodies. Other peptides were recognized by both isotypes. Since these studies suggested that IgG2a may be important in protection against infection, we then investigated some aspects of the role of the 2 IgG2a allotypes, A1 and A2. In retrospective studies of age differences in expression of IgG2a allotypes, no heterozygotes were detected in calves of 60 d old or less, and fewer heterozygotes were detected in calves 61-120 d old than in cattle older than 270 d (P < 0.01). In a subsequent prospective study of the time course of allotype expression, Holstein calves shown to be heterozygotes expressed the IgG2aA1 allotype early but the IgG2aA2 allotype was not usually detected until 3 to 4 mo of age. Thus, both the retrospective and the prospective studies showed age related differences in expression of the IgG2aA1 and A2 allotypes. This could have implication in protection.
Age Factors↗
Workshop summary: local/mucosal immunity.
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Animals↗
Serological response to in vitro-shed antigen(s) of Tritrichomonas foetus in cattle.
We developed a serological assay for detection of (l) an erythrocyte-adhering molecule(s) shed by the bovine venereal pathogen Tritrichomonas foetus and (II) serum antibodies to this antigen(s) in exposed cattle. Sera from exposed and unexposed cattle were tested for their ability to induce complement-mediated lysis of bovine erythrocytes that had been previously incubated overnight at room temperature in pH-adjusted supernatants of T. foetus culture media. Eight of 180 serum specimens from six groups of presumably unexposed cows or heifers showed a positive (> or = 1:2) hemolytic titer (specificity = 95.6%). Thirteen of 14 females in two experimentally infected groups showed a positive hemolytic titer following infection (sensitivity = 94%). In experimentally infected heifers, there was little correlation (r2 = 0.33) between serum hemolytic titers with respect to shed antigen and titers obtained in serum enzyme-linked immunosorbent assays in which whole T. foetus served as the antigen. Serum hemolytic titers rose 3 to 4 weeks sooner than did previously described vaginal mucus immunoglobulin G1 or immunoglobulin A titers with respect to whole-cell antigen or TF1.17 subunit antigen, respectively. Among 14 chronically infected bulls, only 6 (43%) showed a positive hemolytic titer. This study is the first, to our knowledge, to show a specific serological response in the host to an in vitro-shed antigen(s) of T. foetus and suggests a useful diagnostic test for potentially exposed herds.
Administration, Intravaginal↗
Characterization of extracellular proteinases of Tritrichomonas foetus.
Proteinases released from Tritrichomonas foetus into a reducing buffer were characterized because we previously showed that they digested host proteins important in defense of the reproductive tract. These proteinases were shown to be extracellular because cell numbers did not decrease and trichomonads remained motile during their 3.5-hr incubation. Detectable proteinase activity was attributable to enzymes of the cysteine class by spectrophotometric and fluorometric automated assays for peptide substrate specificity. Proteinases from the trichomonad-conditioned buffer were partially purified by bacitracin affinity chromatography. Separation of the eluate on nondenaturing SDS-PAGE gels copolymerized with gelatin, revealed primarily low molecular weight proteinases. Bacitracin-purified T. foetus extracellular cysteine proteinase (TFECP) was separated in a denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel, electroblotted, and a 31-kDa band cut out for amino acid sequencing. Four internal fragments were sequenced, 1 of which contained the highly conserved asparagine region of the cysteine proteinase active site. The combined sequences of these enzyme fragments were 66% and 55% identical to and the corresponding deduced amino acid sequences of 2 T. foetus cysteine proteinase genes (TFCP1 and TFCP2, respectively), which we previously cloned. These results indicate that this enzyme (TFECP) is a distinct cysteine proteinase. The extracellular release of TFECP from T. foetus suggests that it is a potential virulence factor in bovine trichomoniasis.
Amino Acid Sequence↗
Immune and inflammatory responses to reproductive tract infection with Tritrichomonas foetus in immunized and control heifers.
Histopathologic changes and local antibody responses were studied in immunized and control heifers after intravaginal challenge with 10(6) Tritrichomonas foetus. Animals were given 3 intramuscular inoculations of immunoaffinity-purified superficial antigen, TF 1.17, in 2 different adjuvant combinations (incomplete Freund's adjuvant or dextran sulfate plus IFA-8 animals each) or adjuvant alone at 3-wk intervals and were challenged with T. foetus 2 wk later. Histologically, a nonsuppurative endometritis with nodular lymphoid aggregates in the stratum spongiosum was present in 9 of 24 heifers. Twice as many control heifers as immunized had moderate to severe endometritis at 10 wk and the rate of clearance of the organism was significantly faster in immunized than in control heifers. Furthermore, time of clearance was statistically correlated with severity of endometritis at 10 wk postinfection, when necropsies were done (P < 0.02). Because 9-10 wk postinfection is thought to be the critical period for determining fetal loss associated with endometritis, this correlation with early clearance is important to protection against disease. In heifers with moderate to severe infiltration of mononuclear cells in the endometrium, lymphoid nodules and some secondary follicles were detected. In the subgroup of 12 animals from which uterine secretions were collected. IgA antibody responses to antigen were detected by 6 wk in infected animals with increases in mean responses at 8 and 10 wk, but not in uninfected animals. A rationale is presented for consideration of the lymphoid nodules as a possible inductive site for this local antibody response to T. foetus.
Animals↗
Use of an animal model of trichomoniasis as a basis for understanding this disease in women.
Studies of host-parasite relationships in bovine trichomoniasis are presented as a basis for understanding mechanisms of pathogenesis and protection in trichomoniasis in women. The bovine infection is caused by Trichomonas foetus, a trichomonad species closely related to the human pathogen Trichomonas vaginalis. An estrogenized murine model infected with T. vaginalis is useful for the study of virulence factors, but the bovine model has advantages for the study of immunity because T. foetus infection is common in this natural host. In vitro, several virulence factors similar to those of T. vaginalis have been studied, including cysteine proteinases, binding of host cell proteins, and factors involved in attachment to vaginal cells. The biggest advantage of the bovine model, however, is that an efficacious systemic vaccination has been demonstrated to both prevent and treat T. foetus infection. The specificity and isotype of antibodies involved in protection have also been defined. These findings provide encouragement for the development of vaccines against trichomoniasis in humans.
Animals↗
Bovine vaginal antibody responses to immunoaffinity-purified surface antigen of Tritrichomonas foetus.
Bovine trichomoniasis is a prevalent sexually transmitted disease of cattle caused by the protozoan Tritrichomonas foetus. Currently, diagnosis is most often made by culture. In order to provide a faster immunodiagnostic approach, a specific enzyme-linked immunosorbent assay (ELISA) was investigated. A protective surface antigen (TF1.17 antigen) of T. foetus was immunoaffinity purified and used in an ELISA to detect antibodies in vaginal mucus from heifers inoculated with T. foetus. In preliminary studies, antibodies of the immunoglobulin A (IgA) isotype were detected in mucus from all experimentally infected heifers which were tested at 6 weeks postinoculation, whereas IgG1 and IgG2 were not. In addition, IgA responses detected in postinoculation samples were all greater than those detected in preinoculation samples, unlike those detected by a whole-cell antigen ELISA. For these two reasons, IgA antibodies appeared to be useful diagnostically. Further investigation of IgA antibodies used vaginal mucus collected weekly from heifers inoculated intravaginally with 10(2), 10(4), or 10(6) T. foetus organisms. Heifers with positive cultures for T. foetus had similar IgA responses to TF1.17 antigen over the 10 weeks of infection regardless of the initial inoculum dose. This indicates that if the dose is sufficient to establish infection, the magnitude and duration of the immune response are no longer dependent on dose. All of the infected animals receiving all dosages responded with high absorbance values in the IgA anti-TF1.17 antigen ELISA by 6 weeks postinoculation, and all absorbance values remained high at 10 weeks. To determine the duration of the IgA response, four other heifers inoculated with 7 x 10(6) T. foetus organisms were studied through 24 weeks postinoculation. IgA antibody responses to TF1.17 antigen were still high at 24 weeks, even though some heifers cleared the infection several weeks earlier. These results indicate that experimentally infected heifers produced detectable long-lasting IgA responses to TF1.17 antigen in vaginal mucus, which may be useful in a herd test for the diagnosis of bovine trichomoniasis.
Animals↗
A comparative study of bovine and ovine Haemophilus somnus isolates.
Bacterial isolates (including 17 Haemophilus somnus isolates and an H. somnus-like isolate) from asymptomatic or diseased cattle and sheep, were evaluated for markers associated with virulence and host predilection. The isolates were separated into 6 distinct biovariants, 3 for sheep and 3 for cattle, based on reactions in a battery of 21 test media. Three bovine isolates associated with disease caused hemolysis of bovine blood. The rest of the isolates did not hemolyze either bovine or ovine erythrocytes. Protein profiles of all H. somnus isolates were similar with the exception of the major outer membrane proteins (MOMPs). The MOMPs of isolates associated with disease in cattle had a relative molecular weight of approximately 41 kDa compared with 33 kDa for the MOMPs of isolates from asymptomatic cattle. The MOMPs from sheep isolates were either slightly higher or lower than the 41 kDa MOMPs of bovine isolates. Major antigens detected by Western blotting were similar in all isolates except the H. somnus-like isolate. An immunodominant 40 kDa antigen was conserved in all H. somnus isolates. Antibodies to this antigen have previously been found to be protective in cattle and may also be protective for sheep. Marked differences between cattle and sheep isolates were revealed by use of restriction enzyme analysis, which separated the isolates into 12 ribotypes and 15 unique DNA profiles. Thus, cattle and sheep isolates in this collection had distinctive differences in biochemical reactions, MOMP profiles, and DNA analyses. Such differences have potential value for epidemiological studies and may also be used to evaluate host specificity of H. somnus isolates.
Animals↗
Ovine Haemophilus somnus: experimental intracisternal infection and antigenic comparison with bovine Haemophilus somnus.
Experimental infection was produced by two of four isolates of ovine Haemophilus somnus given by intracisternal inoculation into two to three-month-old lambs. Isolate 2041 (originally obtained from a septicemic lamb in Alberta) caused lethal infection in eight of nine lambs, isolate 67p from the prepuce of a normal lamb produced less acute disease in four of nine lambs, and the other two isolates (93p and 1190) caused no detectable disease. Significant lesions were limited to the brain and spinal cord. Purulent meningitis was characteristic but vasculitis or septicemia were not detected, perhaps due to the route of inoculation. Since a difference in virulence was noted among strains, we analyzed surface proteins thought to be virulence factors of bovine H. somnus. Protein profiles of bovine and ovine H. somnus done by sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed similar patterns for virulent bovine isolates and ovine septicemic isolates. Preputial isolates showed a lower molecular mass major outer membrane protein than septicemic isolates. Antigenic analysis revealed that outer membrane proteins p270, p78, p76, p40, and p39 were detected in both ovine and bovine isolates except for 1190, which was probably not a true H. somnus isolate. Thus the preputial and septicemic isolates of ovine H. somnus were similar to bovine H. somnus in pathogenicity and in surface antigens.
Animals↗
Molecular analysis of a gene encoding a serum-resistance-associated 76 kDa surface antigen of Haemophilus somnus.
Haemophilus somnus is a Gram-negative bacterial bovine pathogen which can cause disease or be carried asymptomatically. We previously showed that four serum-sensitive isolates from asymptomatic carriers lacked a 13.4 kb sequence of chromosomal DNA that was present in two virulent serum-resistant strains. We have since sequenced 5 kb of the 13.4 kb fragment from a serum-resistant strain, which contained an open reading frame (ORF) of at least 4.5 kb. From Western blot analysis, the ORF was shown to encode a 76 kDa protein (p76) that co-migrated with a 76 kDa H. somnus surface protein. Both the recombinant and natural p76 reacted with convalescent-phase serum from a cow in an experimental H. somnus abortion study. The translational start site for p76 was identified by deletion analysis of subclones of the 5 kb cloned sequence. The 4.5 kb ORF contained 1.2 kb tandem direct repeats (DRs), with 65% identity between the two repeats at the protein level. The 5' DR (DR1) included the start site for the 76 kDa protein, and DR2 had a flanking inverted repeat, suggestive of an insertion-sequence-like element.
Amino Acid Sequence↗
Immunization of virgin cows with surface antigen TF1.17 of Tritrichomonas foetus.
Protection by surface antigen TF1.17 of Tritrichomonas foetus was investigated because it reacted with a monoclonal antibody which immobilized and mediated complement killing of the organism and prevented adherence to vaginal epithelial cells. This monoclonal antibody was used to demonstrate conservation of the antigen in most strains and to immunoaffinity purify the 50- to 70-kDa glycoprotein antigen. In preparation for immunization studies, the appropriate challenge dose of parasites was determined by intravaginal inoculation of 23 virgin cows (heifers) with 10(2), 10(4), or 10(6) live organisms at the time of estrus. More animals became infected and vaginal infection was maintained at a higher rate (P < 0.005) over 10 weeks for the group that received 10(6) organisms than in the other two groups. Therefore, this dose was used for challenge of immunized animals. Animals immunized with immunoaffinity-purified TF1.17 antigen in incomplete Freund's adjuvant or incomplete Freund's adjuvant plus dextran sulfate cleared the infection more quickly than adjuvant controls (P < 0.005). Isotype-specific enzyme-linked immunosorbent assay with T. foetus antigen showed that serum immunoglobulin G1 (IgG1) and IgG2 antibody responses as well as cervicovaginal mucus IgG1 and IgA antibodies peaked at about the time of clearance of infection in vaccinated animals. Controls developed later cervicovaginal mucus IgA antibody responses as would be expected in a primary local immune response to infection. These results indicate that vaccination with this immunoaffinity-purified surface antigen of T. foetus enhances antibody responses as well as clearance of the parasite from the female reproductive tract.
Animals↗
Conservation of a protective surface antigen of Tritrichomonas foetus.
Bovine trichomoniasis is a sexually transmitted disease caused by the flagellated protozoan Tritrichomonas foetus. A protective surface antigen was previously identified and immunoaffinity purified from T. foetus isolate D1 with cross-reactive monoclonal antibodies (MAbs) TF1.15 and TF1.17 (BonDurant, R. H., R. R. Corbeil, and L. B. Corbeil, Infect. Immun. 61:1385-1394, 1993). This antigen elicited antibody responses in the serum and cervicovaginal mucus of heifers. Thus, it may be useful as an immunodiagnostic reagent as well as a subunit vaccine. Conservation of the antigen in all strains would be crucial for either application. We investigated the conservation of this antigen among 36 isolates of T. foetus from Argentina, Costa Rica, and the United States using MAbs TF1.15 and TF1.17 in an enzyme-linked immunosorbent assay. MAb TF1.17 reacted with 32 of the 36 isolates, whereas MAb TF1.15 reacted with all of the isolates tested. One of the isolates which did not react with MAb TF1.17 (i.e., D1#3) was investigated further by Western blotting (immunoblotting) to determine the reason for the lack of reactivity with one of the two cross-reactive MAbs. The antigenic band that was reactive with MAb TF1.15 had a molecular mass slightly lower than that of the corresponding band from isolate D1, which reacted with both MAbs TF1.15 and TF1.17. Thus, at least a major portion of the antigen appeared to be conserved. This was confirmed in a study of heifers infected with isolate D1#3. The vaginal immunoglobulin A antibodies of these infected heifers reacted with the antigen of isolate D1 that was immunoaffinity purified with MAb TF1.17. Therefore, even though the epitope recognized by MAb TF1.17 was missing in the challenge isolate (D1#3), the heifers developed an immune response to the rest of the molecule. These results indicate that the major portion of the previously described protective antigen is conserved in different isolates of T. foetus. This portion contains the epitope that reacts with MAb TF1.15. Most isolates express the whole antigen, which possesses both TFl.15 and TF1.17 epitopes, but the few isolates that are missing the portion containing the TF1.17 epitope may still elicit an immune reponse to the conserved portion. Thus, the protective surface antigen is promising for use in immunodiagnosis or vaccination against bovine trichomoniasis.
Abortion, Veterinary↗
Two linked genes for outer membrane proteins are absent in four non-disease strains of Haemophilus somnus.
Linked genes encoding two outer membrane proteins (p76 and a family of proteins, p120) of the bovine pathogen, Haemophilus somnus, were investigated. The p120 group was previously shown to have immunoglobulin-binding activity and to react with polyclonal antiserum specific for a 270 kDa antigen (p270) which also had immunoglobulin Fc-binding activity. By Western blotting we showed that the p76 antigen also reacted with this antiserum. The p270, p120, and p76 antigens were undetectable in four serum-sensitive isolates from asymptomatic carriers but were present in the two serum-resistant virulent strains tested. Genes for p120 and p76 were subcloned on non-overlapping pUC plasmids from a cosmid (pHS1) originally cloned from a serum-resistant strain. In Escherichia coli, plasmid pHS138 expressed p76, while the p120 antigens were produced by pHS140. Southern blots of DNA from the above six strains of H. somnus using probes derived from pHS1 subclones showed that a 13.4 kb sequence was missing from the four serum-sensitive strains, but not the two serum-resistant strains. This segment included most of the insert in pHS138 and all of the pHS140 insert. The data indicate that p76 and the p120 proteins are absent from serum-sensitive strains because the coding sequences are missing, raising the possibility of insertion of these genes into the chromosome of both serum-resistant strains, or deletion from the four serum-sensitive strains.
Antigens, Bacterial↗