Effect of Histophilus somni on Heart and Brain Microvascular Endothelial Cells.

Histophilus somni is a pathogenic gram-negative bacterium responsible for pneumonia and septicemia in cattle. Sequelae include infectious thrombotic meningoencephalitis (ITME), myocarditis, arthritis, and abortion. These syndromes are associated with widespread vasculitis and thrombosis, implicating a role for endothelium in pathogenesis. Histopathologic and immunohistochemical investigation of 10 natural cases of bovine H. somni myocarditis and 1 case of ITME revealed intravascular H. somni in large biofilm-like aggregates adherent to the luminal surface of microvascular endothelium. Ultrastructurally, bacterial communities were extracellular and closely associated with degenerating or contracted endothelial cells. Histophilus somni was identified by bacterial culture and/or immunohistochemistry. Western blots of the bacterial isolates revealed that they expressed the immunodominant protective 40 kDa OMP and immunoglobulin-binding protein A (IbpA) antigens. The latter is a large surface antigen and shed fibrillar antigen with multiple domains. The cytotoxic DR2Fic domain of IbpA was conserved as demonstrated by polymerase chain reaction. Treatment of endothelial cells in vitro with IbpA in crude culture supernatants or purified recombinant GST-IbpA DR2Fic (rDR2) cytotoxin induced retraction of cultured bovine brain microvascular endothelial cells. By contrast, no retraction of bovine endothelium was induced by mutant rDR2H/A with an inactive Fic motif or by a GST control, indicating that the cytotoxic DR2Fic motif plays an important role in endothelial cell retraction in vasculitis. The formation of biofilm-like aggregates by H. somni on bovine microvascular endothelium may be fundamental to its pathogenesis in heart and brain.

Bovine respiratory syncytial virus and Histophilus somni interaction at the alveolar barrier.

Our previous studies showed that Histophilus somni and bovine respiratory syncytial virus (BRSV) act synergistically in vivo to cause more severe bovine respiratory disease than either agent alone causes. Since H. somni surface and secreted immunoglobulin binding protein A (IbpA) causes retraction of bovine alveolar type 2 (BAT2) cells and invasion between BAT2 cells in vitro, we investigated mechanisms of BRSV-plus-H. somni infection at the alveolar barrier. BRSV treatment of BAT2 cells prior to treatment with IbpA-rich H. somni concentrated culture supernatant (CCS) resulted in increased BAT2 cell rounding and retraction compared to those with either treatment alone. This mimicked the increased alveolar cell thickening in calves experimentally infected with BRSV followed by H. somni compared to that in calves infected with BRSV or H. somni alone. BRSV-plus-H. somni CCS treatment of BAT2 cells also enhanced paracellular migration. The effect of matrix metalloproteinases (MMPs) was investigated as well because microarray analysis revealed that treatment with BRSV plus H. somni synergistically upregulated BAT2 cell expression of mmp1 and mmp3 compared to that in cells treated with either agent alone. Enzyme-linked immunosorbent assay (ELISA) confirmed that MMP1 and MMP3 protein levels were similarly upregulated. In collagen I and collagen IV (targets for MMP1 and MMP3, respectively) substrate zymography, digestion was increased with supernatants from dually treated BAT2 cells compared with those from singly treated cells. Enhanced breakdown of collagen IV in the basal lamina and of fibrillar collagen I in the adjacent interstitium in the dual infection may facilitate dissemination of H. somni infection.

Antibodies to Trichomonas vaginalis surface glycolipid.

BACKGROUND: Human trichomoniasis is the most common non-viral sexually transmitted disease, yet immune responses are not well studied. METHODS: Since the Trichomonas vaginalis lipophosphoglycan (TvLPG) is an important virulence factor, a bank of eight monoclonal antibodies was generated to define the antigen in clinical isolates. The TvLPG-specific antibody response of women who were culture positive (n=33) or negative (n=33) for T vaginalis infection was determined by isotype-specific ELISA. RESULTS: The bank of monoclonal antibodies reacted with conserved surface TvLPG epitopes in 27 isolates from pregnant women at their first prenatal visit. Conserved TvLPG epitopes were shown to be surface exposed by immunofluorescence. Sera collected from the same patients at the same time were assayed for specific antibodies. Serum and vaginal secretions from 33 T vaginalis-positive women had statistically higher IgG anti-TvLPG levels than age-matched and race-matched negative controls in the same clinical study (p<0.01). Vaginal IgA anti-TvLPG levels of the women with trichomoniasis were almost significantly higher than controls (p=0.055). Infected women with normal pregnancies had significantly higher vaginal IgG anti-TvLPG values than infected women with adverse outcomes of pregnancy. CONCLUSIONS: These antibody responses show that infected women can respond to the conserved TvLPG antigen. Since antibodies to trichomonad surface LPG protect in a bovine model of trichomoniasis, the role of these antibodies in the human disease should be investigated.

Diagnostic exercise: Myocarditis due to Histophilus somni in feedlot and backgrounded cattle.

A backgrounding operation for calves in Wyoming identified a disease syndrome presenting as lethargy, fever, and death between November and January each year. An unfixed heart was submitted for examination, along with samples of lung. There was focal red discoloration in papillary muscle of the left ventricular myocardium. Histologically, the lesion corresponded to acute necrotizing myocarditis with myriad intravascular and intralesional gram-negative coccobacilli. Histophilus somni was detected by bacterial culture and immunohistochemistry. Focal myocarditis due to H. somni occurs in fall-placed cattle in western provinces and states of North America, and it can be an appreciable source of death loss. Gross lesions are readily detected in affected hearts. The presence of such changes in papillary muscles of left ventricular myocardium in feedlot or backgrounded cattle should prompt a differential diagnosis of H. somni myocarditis.

Comparative histopathology and antibody responses of non-Tritrichomonas foetus trichomonad and Tritrichomonas foetus genital infections in virgin heifers.

The potential pathogenicity of non-Tritrichomonas foetus trichomonads (NTfTs) recently isolated from the prepuce of virgin bulls is not known. The purpose of this study was to determine the ability of these NTfTs to cause disease in the female reproductive tract relative to T. foetus. Forty-four virgin heifers were experimentally infected intravaginally with either one of two NTfTs (Pentatrichomonas hominis or Tetratrichomonas spp.), T. foetus, or sterile media and cultured weekly from 0 time until slaughter at 8 weeks. Serum and vaginal antibody responses during infection were assessed, and the reproductive tracts were histologically examined, scored, and compared based on numbers of neutrophils, eosinophils, lymphocytes, and plasma cells as well as the qualitative appearance of the reproductive tract. The NTfTs did not persist in the reproductive tract, while T. foetus persisted for at least 6-8 weeks. Further, no vaginal IgA response to infection was found in NTfT-infected and control heifers, but a vaginal IgA response was present in the T. foetus-infected group. Heifers infected with NTfT or controls showed little mucosal inflammatory response compared to T. foetus-infected heifers. Among the trichomonads studied, persistent infection by T. foetus alone seems responsible for uterine inflammatory lesions usually associated with pregnancy loss. The NTfTs studied in this work only transiently infected the vagina and were associated with strictly mild inflammatory changes, which probably do not cause significant disease, i.e., pregnancy loss.

Induction of immunity against lethal Haemophilus influenzae type b infection by Escherichia coli core lipopolysaccharide.

Efforts to prevent Haemophilus influenzae type b (HIB) infections in infancy have been hampered by the low immunogenicity of capsular polysaccharide vaccines in children younger than 18 mos. In searching for alternate immunogens, we have studied the protective potential of polysaccharide-poor, lipid-rich endotoxin (LPS) core in experimental HIB infections. Because all gram-negative bacteria have similar LPS core structures, we were able to use as vaccine the J5 mutant of Escherichia coli 0111, the LPS of which consists only of core components, and thus to avoid problems in interpretation arising from vaccine contamination with non-LPS HIB immunogens. Mice were given graded inocula of HIB and developed lethal infection analogous to human HIB disease when virulence was enhanced with mucin and hemoglobin. After active immunization with heat-killed E. coli J5, 40/50 (80%) of infected mice survived, compared with 14/50 (28%) of saline-immunized controls (P less than 0.005). Passive immunization with rabbit antiserum against E. coli J5 prevented lethal HIB infection when administered 24 or 72 h before or 3 h after infection. This protection was abolished by adsorption of antiserum with purified J5 LPS, with survival reduced from 14/24 to 0/24 (P less than 0.005). Furthermore, rabbit antiserum to purified J5 LPS gave just as potent protection against death as antiserum to whole J5 cells. These studies demonstrate that immunity to core LPS confers protection against experimental murine HIB infection and provide the framework for a new approach to prevention of human disease from HIB.

Evaluation of surface antigen TF1.17 in feline Tritrichomonas foetus isolates.

Tritrichomonas foetus (T. foetus) is a flagellated protozoa that infects the distal ileum and proximal colon of domestic cats, as well as the urogenital tract of cattle. Feline trichomonosis is recognized as a prevalent cause of chronic diarrhea in cats worldwide. The suspected route of transmission is fecal-oral, with cats in densely crowded environments at highest risk for infection. Thus, the recommended strategy for minimizing spread of infection is to identify and isolate T. foetus-positive cats from the general population. Rapid identification of infected cats can be challenging due to the inability to accurately and quickly detect the organism in samples at point of care facilities. Thus, identification of targets for use in development of a novel diagnostic test, as well as a vaccine or therapy for T. foetus infection is a significant area of research. Despite a difference in organ tropism between T. foetus genotypes, evidence exists for conserved virulence factors between feline and bovine T. foetus. The bovine T. foetus surface antigen, TF1.17, is an adhesin that is conserved across isolates. Vaccination with the purified antigen results in amelioration of cytopathogenicity and more rapid clearance of infection in cattle. We previously showed that three feline isolates of T. foetus were positive for TF1.17 antigen so we further hypothesized that TF1.17 is conserved across feline T. foetus isolates and that this antigen would represent an attractive target for development of a novel diagnostic test or therapy for feline trichomonosis. In these studies, we used monoclonal antibodies previously generated against 1.15 and 1.17 epitopes of the bovine T. foetus TF1.17 antigen, to evaluate for the presence and role of TF1.17 in the cytopathogenicity of feline T. foetus. A previously validated in vitro co-culture approach was used to model feline T. foetus infection. Immunoblotting, immunofluorescence assays, and flow cytometric analysis confirmed the presence and surface localization of antigen TF1.17 across all feline T. foetus isolates tested. Antigen TF1.17 was notably absent in the presumably nonpathogenic intestinal trichomonad, Pentatrichomonas hominis, a parasite that can be confused microscopically with T. foetus. Similar to bovine trichomoniasis, TF1.17 was found to promote T. foetus adhesion to the intestinal epithelium. These results support further characterization and development of the TF1.17 antigen as a possible target for the diagnosis and prevention of feline T. foetus infection.

Diatoms and diatomaceous earth as novel poultry vaccine adjuvants.

Diatoms are single cell eukaryotic microalgae; their surface possesses a porous nanostructured silica cell wall or frustule. Diatomaceous earth (DE) or diatomite is a natural siliceous sediment of diatoms. Since silica has been proved to have adjuvant capabilities, we propose that diatoms and DE may provide an inexpensive and abundant source of adjuvant readily available to use in livestock vaccines.In a first experiment, the safety of diatoms used as an adjuvant for in-ovo vaccination was investigated. In a second experiment, we assessed the humoral immune response after one in-ovo vaccination with inactivated Newcastle Disease Virus (NDV) and DE as adjuvant followed by 2 subcutaneous boosters on d 21 and 29 of age. In both experiments, results were compared to Freund's incomplete adjuvant and aluminum hydroxide.No detrimental effects on hatchability and chick quality were detected after in-ovo inoculation of diatoms and DE in experiments 1 and 2 respectively. In experiment 2 no humoral responses were detected after the in-ovo vaccination until 29 d of age. Seven d after the second subcutaneous booster an antibody response against NDV was detected in chickens that had received vaccines adjuvanted with Freund's incomplete adjuvant, aluminum hydroxide, and DE. These responses became significantly higher 10 d after the second booster. Finally, 15 d after the second booster, the humoral responses induced by the vaccine with Freund's incomplete adjuvant were statistically higher, followed by comparable responses induced by vaccines containing DE or aluminum hydroxide that were significantly higher than DE+PBS, PBS+INDV and PBS alone. From an applied perspective, we can propose that DE can serve as a potential adjuvant for vaccines against poultry diseases.

Serum, uterine, and vaginal mucosal IgG antibody responses against Tritrichomonas foetus after administration of a commercial killed whole T foetus vaccine in beef cows.

The objective of this study was to determine the level and duration of IgG antibodies induced against killed whole Tritrichomonas foetus and T foetus-purified surface antigen (TF1.17) in serum, vaginal, and uterine secretions after systemic immunization of beef cows with a vaccine containing killed whole T foetus. Twenty nonpregnant beef cows were randomly assigned to vaccine or control groups as follows: Vaccine (n = 10): cows received 2 mL of a commercial vaccine containing killed whole T foetus subcutaneously and a 2-mL booster 2 weeks later. Control (n = 10): cows received 2 mL of sterile saline on the same schedule. Vaginal secretions and blood samples were collected on Days 0, 8, 15, 22, 29, 36, 43, 50, 60, 75, 89, 110, 146, and 182 relative to day of primary vaccination. Uterine flush fluid was collected on Days 0, 15, 29, and 43 after the day of primary vaccination. Samples were assayed for IgG antibodies to the killed whole T foetus and surface antigen TF1.17 using enzyme-linked immunosorbent assay. Serum whole T foetus-specific IgG levels were significantly increased (between Days 15 and 182) following vaccination with T foetus or with saline. No differences between vaccinates and controls in uterine responses to whole-cell antigen were detected. Serum anti-TF1.17 IgG responses to vaccination were significantly higher than Day 0 throughout the immunization period (P < 0.001) and were higher than responses in control animals on each day post immunization through Day 146 (P < 0.001). A significant rise in TF1.17-specific IgG levels was observed in vaginal and uterine fluids from Day 15 post vaccination compared to the Day 0 levels. These levels remained significantly elevated in vaginal and uterine fluids through Days 75 (P < 0.05) and 43 (P < 0.001) after primary vaccination, respectively. Antibody levels in serum, vaginal, and uterine secretions against TF1.17 remained low in the control group throughout the study. In conclusion, vaccination of beef cows with a commercial vaccine containing T foetus induced significant increase in the levels of IgG to the T foetus TF1.17 surface antigen in serum, vaginal secretions, and uterine fluid, which remained elevated through Days 43, 75, and 182 in uterine fluids, vaginal secretions, and serum, respectively. Since purified TF1.17 antigen has been shown to protect against experimental T foetus infection in heifers, the vaccine-induced TF1.17-specific IgG response is likely to be important in the prevention of trichomoniasis in beef cattle.

Histophilus somni Stimulates Expression of Antiviral Proteins and Inhibits BRSV Replication in Bovine Respiratory Epithelial Cells.

Our previous studies showed that bovine respiratory syncytial virus (BRSV) followed by Histophilus somni causes more severe bovine respiratory disease and a more permeable alveolar barrier in vitro than either agent alone. However, microarray analysis revealed the treatment of bovine alveolar type 2 (BAT2) epithelial cells with H. somni concentrated culture supernatant (CCS) stimulated up-regulation of four antiviral protein genes as compared with BRSV infection or dual treatment. This suggested that inhibition of viral infection, rather than synergy, may occur if the bacterial infection occurred before the viral infection. Viperin (or radical S-adenosyl methionine domain containing 2--RSAD2) and ISG15 (IFN-stimulated gene 15--ubiquitin-like modifier) were most up-regulated. CCS dose and time course for up-regulation of viperin protein levels were determined in treated bovine turbinate (BT) upper respiratory cells and BAT2 lower respiratory cells by Western blotting. Treatment of BAT2 cells with H. somni culture supernatant before BRSV infection dramatically reduced viral replication as determined by qRT PCR, supporting the hypothesis that the bacterial infection may inhibit viral infection. Studies of the role of the two known H. somni cytotoxins showed that viperin protein expression was induced by endotoxin (lipooligosaccharide) but not by IbpA, which mediates alveolar permeability and H. somni invasion. A naturally occurring IbpA negative asymptomatic carrier strain of H. somni (129Pt) does not cause BAT2 cell retraction or permeability of alveolar cell monolayers, so lacks virulence in vitro. To investigate initial steps of pathogenesis, we showed that strain 129Pt attached to BT cells and induced a strong viperin response in vitro. Thus colonization of the bovine upper respiratory tract with an asymptomatic carrier strain lacking virulence may decrease viral infection and the subsequent enhancement of bacterial respiratory infection in vivo.

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.

Non-immune immunoglobulin binding by "Haemophilus somnus".

In-vitro culture of Haemophilus somnus in liquid or solid media supplemented with bovine blood or serum resulted in non-immune binding of immunoglobulin (Ig) by the organism. This binding was independent of the antigen-combining site of the Ig molecule, since binding of an IgG preparation specific for the hapten dinitrophenol was unaffected by the presence of the homologous antigen. Quantitative comparison of the binding of Ig fragments Fab and Fc demonstrated that the non-immune binding occurred in the Fc region of bovine IgG. The isotypes of Ig that became bound to H. somnus included both bovine IgG subclasses (IgG1 and IgG2), which were bound equally, and bovine IgM.

Immunological characterization of the major outer membrane protein of Haemophilus somnus.

Antigens and molecular mass diversity of the Haemophilus somnus major outer membrane protein (MOMP) were investigated. The molecular mass of the MOMP of 53 strains of H. somnus varied from 43 to 33 kDa and four MOMP MAb reactivity patterns were detected in immunoblot analysis and immunodot assay. The molecular mass and MAb reactivity data were used for preliminary grouping of H. somnus strains. Disease strains fell into groups 1 and 3, including two of three Group 3 subgroups, whereas strains from asymptomatic carriers were found in all the four groups and three subgroups. Immunoblot analysis with convalescent phase serum showed strain specific reactivity with MOMPs from three isolates used to reproduce disease in cattle. The reaction with the MOMP was only detectable at dilutions of 1:100 or less, whereas the same convalescent sera showed strong reactivity at dilutions of 1:1000 (or more) with other H. somnus antigens. The data suggest that the bovine immune response to the MOMP during infection is weak and is directed to antigenically variable determinants in a strain-specific manner. This may be important in evaluating the role of the antibody response to MOMPs in protective immunity.

Teat skin normal flora and colonization with mastitis pathogen inhibitors.

Isolates of bacteria from normal teats were used to attempt colonization of teats of dry cows or neonatal calves. Isolates for inoculation were chosen on the basis of ability to inhibit mastitis pathogens in vitro, with the ultimate goal of in vivo inhibition of mastitis pathogens at the teat surface. Three bacterial normal flora isolates (Corynebacterium xerosis, Bacillus sp. and Aerococcus viridans) persisted less than 10 days on the teats of dry cows. The fourth isolate, Staphylococcus hominis 1, was studied in greatest detail because studies characterizing the normal teat flora showed staphylococci to be the predominant flora. The S. hominis 1 isolated used for inoculation was an inhibitor of Gram-positive mastitis pathogens. It was a biotype not found on these teats prior to inoculation, thus facilitating identification of the inoculated isolate on sequential sampling. Colonization of newborn calves, before other bacterial flora became established, resulted in recovery of inoculated S. hominis 1 for an average of 51 days or longer. On dry cow teats it was detected for up to 28 days. On several occasions the inoculated S. hominis 1 was found in pure culture. Since many new infections occur during the dry period, the colonization of dry cow teats with S. hominis 1 organisms inhibitory for Gram-positive pathogens should be tested as an adjunct to other methods of mastitis prevention.

Cross-reactive antibody in immunity to colisepticemia in calves.

Cattle were immunized with a uridine diphosphate galactose epimerase deficient mutant of Escherichia coli to prepare antiserum cross-reactive with different serotypes of E. coli. Hypogammaglobulinemic calves were given bovine anti-J5 serum before oral challenge with virulent E. coli derived from a septicemic calf. Passively immunized calves had delayed and decreased bacteremia compared with calves given saline before challenge. Calves given antiserum also lived longer than control calves. A second experiment using ampicillin and antibody to treat colisepticemia also showed increased survival in anti-serum-treated calves. Decreased bacteremia was probably not due to the killing of the challenge strain by antibody and complement, as the strain was serum-resistant. However, anti-J5 serum did increase phagocytosis of the challenge strain of E. coli (JL9) by bovine neutrophils. Thus, partial protection by antiserum was probably due to increased clearance of bacteria as well as neutralization of endotoxin.

Antibodies as effectors.

Antibodies are critical in protection against extracellular microbial pathogens. Although antibodies also play a role in transplant/tumor rejection and in autoimmune disease, this paper focuses on defense against bovine infections. Effector mechanisms of different bovine isotypes, subisotypes and allotypes are discussed. The importance of antigen specificity is also stressed.

Binding of bovine IgG2a and IgG2b allotypes to protein A, protein G, and Haemophilus somnus IgBPs.

Immunoglobulin binding proteins (IgBPs) are thought to be virulence factors which enable pathogens to evade the host's immune response. Since bovine IgG2 is important in protection against pyogenic infections, the binding characteristics of Staphylococcus aureus protein A (PrA), streptococcal protein G (PrG), or Haemophilus somnus high molecular weight IgBPs to the two bovine IgG2 allotypes were examined. For PrA or PrG binding of IgG2, guinea pig red blood cells coated with specific IgG2a or IgG2b antibodies were used in a competitive binding inhibition assay with unlabeled and horseradish peroxidase-labeled PrA or PrG. To determine which sizes of H. somnus. IgBPs bind to the two IgG2 allotypes, immunoblots with H. somnus culture supernatant were probed with anti-DNP IgG2a and IgG2b. This detects only Fc binding because anti-DNP does not cross-react with H. somnus antigens. Both IgG2 allotypes bound equally well to PrA and PrG. However, IgG2b but not IgG2a bound to H. somnus high molecular weight IgBPs. The lack of differential binding of bovine IgG2 allotypes to PrA and PrG means that these IgBPs can be considered to be unbiased reagents in assays for detection of bovine IgG2 or for immunoaffinity purification. The differential binding of H. somnus IgBPs to the IgG2 allotypes indicates that animals having one allotype may be more resistant to H. somnus infection than animals having the other allotype.

Differential complement activation by bovine IgG2 allotypes.

Immunoglobulin allotypes and complement (C) are known to be related to susceptibility to infection. Because bovine IgG2 is important in resistance to pyogenic infections and because its two allotypes, IgG2a and IgG2b, differ in sequence in the CH1, hinge, CH2, and CH3 regions, we tested the ability of these allotypes to initiate the bovine C cascade. Bovine IgG2a and IgG2b were standardized according to specific anti guinea pig red blood cell (GPRBC) ELISA activity using anti IgG2 reagents shown essentially unbiased for allotype. Complement activating activity of the allotypes was quantitated in a GPRBC lysis assay. With this system, IgG2b consistently had more than twice the activity in bovine C mediated lysis as compared with IgG2a. The fact that both EDTA and EGTA/Mg almost completely inhibited C mediated lysis of GPRBCs indicated that lysis was due to the classical pathway. Since antibody usually activates C by the classical pathway, this supports the supposition that activation was by the IgG2-GPRBC complexes. Flexibility analyses showed that IgG2b had a more rigid hinge than IgG2a, perhaps partially explaining the greater efficiency of IgG2b in C activation. Other mechanisms may include differences in glycosylation and in the amino acid at position 332. The difference in ability to activate C may mean that animals of the IgG2a allotype could be more susceptible to infection with extracellular pyogenic pathogens which are killed by C or by phagocytes after opsonization with IgG2 and C.

Degradation of bovine complement C3 by trichomonad extracellular proteinase.

Bovine trichomoniasis is a local infection of the reproductive tract making interaction with mucosal host defenses crucial. Since the parasite is susceptible to killing by bovine complement, we investigated the role of the third component of complement (C3) in host parasite interactions. Bovine C3 was purified by anionic and cationic exchange chromatography. The purified protein was characterized by immunoreactivity, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and peptide sequencing of the amino terminus of the beta chain. When purified bovine C3 was incubated for varying time periods with trichomonad extracellular proteinases, SDS-PAGE gels revealed digestion of the alpha chain to small fragments. Such degradation in vivo would prevent formation of C3b and completion of the complement cascade, resulting in evasion of killing. To evaluate the relevance of this data, we determined whether C3 was present in bovine genital secretions. With a quantitative ELISA assay, C3 could be demonstrated in both uterine and vaginal washes. To our knowledge, this is the first demonstration of bovine C3 in genital secretions. The C3 concentration increased significantly in vaginal secretions by 8 and 10 weeks in heifers infected with Tritrichomonas foetus. An increase was also seen in uterine secretions of infected heifers, but sample numbers were insufficient for statistical analysis. Transcription of the major extracellular cysteine proteinase (TFCP8) was demonstrated in T. foetus cells from uterine secretions of infected heifers by RT-PCR and Southern blotting. The results indicate that C3 may be important in genital defense and that trichomonad extracellular proteinases may play a role in evasion of complement-mediated killing.

Ultrastructure of cyclic changes in the murine uterus, cervix, and vagina.

The regional and cyclic changes in the murine genital epithelium were studied by transmission and scanning electron microscopy to provide a morphological standard to serve as a basis for investigation of host-parasite relationships in genital infections. Thus, we examined not only mucosal epithelial cell changes, but also surface mucus, normal flora and inflammatory cells. Ultrastructurally, at proestrus/estrus, we found uterine and most cervical epithelial cells covered with microvilli overlaid with mucus-like secretions and evidence of internal secretory activity. There was little normal flora anywhere in the tract. At early metestrus, we found squamous cervicovaginal epithelial cells with low discontinuous microrugae, extensive normal flora and many neutrophils beginning to migrate through the epithelium. The flora and neutrophils could explain the relative lack of susceptibility to infection at that time. At diestrus the appearance of a newly regenerated epithelium and lack of normal flora suggested that initiation of infection could occur at this stage; however, the presence of large numbers of neutrophils ready to phagocytize invading bacteria indicated a deterrent to infection. This study of cyclic changes in flora, mucus, neutrophils and epithelial cells provided ultrastructural evidence to support an earlier hypothesis that the greatest susceptibility to gonococcal infection in mice occurred at proestrus/estrus.