Possible presence of a capsule in Branhamella catarrhalis.

Clinical isolates of Branhamella catarrhalis from patients with respiratory infections were used in this study. Electron microscopic observation after treating Branhamella catarrhalis with immune serum and ruthenium red revealed the capsule. In the phagocytosis test, most organisms were not ingested by human polymorphonuclear neutrophils in the presence of normal rabbit serum (NRS), while organisms were primarily cell associated and apparently ingested in the presence of immunized rabbit serum (IRS). The capsule may be one of the virulence factors in this bacteria. This study demonstrates the possible presence of a capsule in Branhamella catarrhalis.     

Inhibition of immune precipitation by complement

Normal human complement serum (NHS) inhibited precipitin reactions between tetanus toxoid and human or rabbit anti-tetanus toxoid IgG antibody, between bovine serum albumin (BSA) and rabbit anti-BSA IgG antibody, and between hen egg albumin and rabbit anti-egg albumin IgG antibody. Ethylene-diaminetetraacetic acid (EDTA) prevented this inhibition. Mg-ethyleneglycol-bis(aminoethyl)-tetra-acetic acid-(EGTA) also prevented the inhibition except with lower concentrations of antibody and antigen. Therefore, the inhibition of immune precipitation seemed to occur mainly through the classical pathway of complement activation. The alternative pathway was usually dispensable, but it augmented the inhibition. Guinea pig complement serum (NGS) was less effective than NHS in inhibiting immune precipitation. Guinea pig serum deficient in C4 (C4DGS) did not inhibit the immune precipitation. Mouse complement serum was effective for inhibiting precipitation, and C5-deficient serum was as effective as normal serum. Therefore, the inhibition of immune precipitation is considered to occur by activation of complement up to the step of C3. The size of the soluble immune complexes formed in the presence of NHS varied depending on the concentrations of antibody and antigen, even when the ratio of antigen to antibody was constant. On incubation at 37 degrees C immune precipitation was inhibited by 1/2 dilution of NHS for 2 to 3 hr and then gradually increased to the level in the absence of complement. When the immune complexes were formed in the presence of serum containing complement, fragments of C4 and C3 were incorporated into the soluble immune complexes. The C3 fragments incorporated into the soluble complexes were C3b, iC3b, C3c, and C3d, some of which were bound covalently with heavy chains of IgG antibody molecules. Some of the covalent linkages between C3 fragments and IgG seemed to be destroyed by alkali treatment, but not by hydroxylamine treatment. The formation of covalent bonds between IgG and C3 and probably C4 was essential for inhibition of immune precipitation, because inhibitors of their formation, such as putrescine, cadaverine, and salicylhydroxamic acid, effectively prevented the inhibition of precipitation. When antigen and antibody reacted in the presence of mixtures of various combinations of isolated complement components, C1, C4, C2, and C3 showed maximal inhibition of immune precipitation, whereas factors I and H had little effect.     

Serological Diagnosis of Classical Swine Fever

Antibody levels in post-infection sera from a pig inoculated with a low virulent strain of classical swine fever virus (Hannover 62) and in sera from two pigs inoculated with another low virulent strain (Spielbach 66) and from an in-contact pig were assayed by complement fixation and immunofluorescence using classical swine fever virus (ALD strain) and bovine virus diarrhoea virus (UG 59 strain) as antigens. The complement fixation test used was modified by addition of a preparation of porcine Glq to the complement and by mercaptoethanol treatment of the immune serum before use. The mercaptoethanol treatment of the immune serum resulted in complete elimination of a haemolytic prozone often seen with porcine immune sera. In the sera from the inoculated animals complement-fixing antibodies appeared earlier than neutralizing antibodies. A few weeks after inoculation there was a correlation between the presence of complement-fixing and neutralizing antibodies. During the entire observation period of 13 weeks it was not possible to demonstrate complement-fixing or neutralizing antibodies in serum from a pig exposed to infection by contact with the two pigs inoculated with the Spièlbach 66 strain of classical swine fever virus.     

Lytic effects of normal serum on isolated postonchospheral and metacestode stages of Taenia taeniaeformis

Postonchospheral stages of Taenia taeniaeformis liberated from rat livers by enzymatic digestion at 1 to 10 days postinfection (DPI) and metacestodes dissected from infected livers at 22, 34, and 69 DPI were exposed in vitro to immune rat serum (IRS) and to normal serum from rats (NRS), human beings (NHS), or guinea pigs (NGS). The onset of rapid and destructive tegumental changes in all organisms exposed to any of the sera was demonstrated to be complement-dependent because the reaction was: (a) inhibited by treatment of serum at 56 C for 30 min; (b) inhibited by prior incubation of serum with zymosan or with complement-fixing, soluble products derived from larvae of T. taeniaeformis maintained in vitro (IVP); and (c) abolished by the addition of EDTA. Lytic effects occurred on exposure to agammaglobulinemic sheep serum, and lysis in the presence of IRS and NRS was shown to result in consumption of available hemolytic complement. Surface changes consisted of vesiculation in the microvillar or microthrix layers followed by sloughing of the tegument, eventually leading to collapse of the cystic bladder and cessation of flame cell activity, or, in the case of early postonchospheral forms, complete disintegration of the organism. When IVP was added to NHS, reduction of hemolytic complement activity was associated with the electrophoretic conversion of C3, and Factor B, but there was little or no consumption of C1. The observations support the hypothesis that complement-mediated effector mechanisms must be counteracted to ensure survival of parasites in vivo, and that the capacity for release of soluble nonspecific complement-fixing factors by taeniid larvae may have an important role to play in this process.     

Effect of anti-sperm antibody on the in vitro development of rat embryos

Since we previously proved that the fertilized rat eggs in early developmental stage have antigen(s) cross-reacting to spermatozoa, the effect of antibody to spermatozoa on the cleavage of fertilized rat eggs was examined in vitro. Fertilized eggs from Fisher rats in the morula stage were cultured in vitro for 15 to 39 hr in the medium containing antibody to rat spermatozoa and rabit complement, and the developmental rates of morulae to blastocysts were compared with those cultured in the presence of either antibody or the complement alone. When rat morulae were cultured in the medium containing rabbit complement and IgG from rabbit antiserum to rat spermatozoa (heteroantibody) or from rat antiserum to rat spermatozoa (isoantibody), the development of moralae to blastocysts was markedly suppressed, whereas those cultured in the medum containing rabbit complement and IgG from the control rabbit serum or rabbit antibody IgG to rat spermatozoa alone without complement normally developed to the blastocysts. These results indicate that the antibody to spermatozoa in presence of complement can impair the in vitro development of fertilized rat eggs.     

The binding of complement component C3 to antibody-antigen aggregates after activation of the alternative pathway in human serum.

Preformed immune aggregates, containing antigen and either IgG (immunoglobulin G) or F(ab')2 rabbit antibody, were incubated with normal human serum under conditions allowing activation of only the alternative pathway of complement. Both the IgG and F(ab')2 immune aggregates bound C3b, the activated form of the complement component C3, in a similar manner, 2-3% of the C3 available in the serum being bound to the aggregates as C3b, and the rest remaining in the fluid phase as inactive C3b or uncleaved C3. It was found that the C3b was probably covalently bound to the IgG in the aggregates, since C3b-IgG complexes could be demonstrated on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, after repeated washing with buffers containing high salt or boiling under denaturing conditions. Incubation of the C3b-antibody-antigen aggregates in buffers known to destroy ester linkages had little effect on the C3b-IgG complexes, which suggested that C3b and IgG might be linked by an amide bond. Two main types of C3b-IgG complexes were found that had apparent mol.wts. of 360000 and 580000, corresponding to either one to two C3b molecules respectively bound to one molecule of antibody. On reduction of the C3b-IgG complexes it was found that the beta-chain, but not the alpha'-chain, of C3b was released along with all the light chain of IgG but only about half or less of the heavy chain of IgG. These results indicate that, during activation of the alternative pathway of complement by immune aggregates containing IgG antibody, the alpha'-chain of C3b may become covalently bound at one or two sites in the Fd portion of the heavy chain of IgG.     

Specific antibody promotes opsonization and PMN-mediated killing of phagocytosis-resistant Enterococcus faecium

Many clinical isolates of Enterococcus faecium are resistant to neutrophil (PMN)-mediated phagocytosis and killing in the presence of normal human serum. We have now examined the ability of specific polyclonal rabbit antibodies to promote opsonization and killing of phagocytosis-resistant E. faecium. Immune rabbit serum generated against formalin-killed E. faecium TX0016, a phagocytosis-resistant strain, markedly promoted binding of TX0016 organisms to PMNs and PMN-mediated killing. These effects were dramatically reduced by (a) adsorption of immune serum with E. faecium TX0016, but not by adsorption with a strain of E. faecium susceptible to phagocytosis, and (b) incubation of immune serum with carbohydrate purified from TX0016, but not by incubation with a surface protein extract from TX0016. IgG purified from immune serum was unable by itself to promote bacterial binding to PMNs. However, specific IgG was able to promote binding to PMNs and PMN-mediated killing in the presence of normal human serum as a complement source, as were F(ab')(2) and Fab fragments produced from it, and the alternative pathway of complement was sufficient to promote IgG- and F(ab')(2)-mediated opsonization. PMN complement receptor type 3, but not complement receptor type 1, was involved in bacterial binding to PMNs induced by the combination of F(ab')(2) fragments and normal human serum. These results suggest that opsonization by antibodies potentially directed against bacterial carbohydrate, in conjunction with complement activation, has an important role in the host defense against phagocytosis-resistant E. faecium.     

Endogenous vasoactive intestinal peptide (VIP) regulates somatostatin secretion by cultured fetal rat cerebral cortical and hypothalamic cells.

To determine the possible physiological role of endogenous vasoactive intestinal peptide (VIP) in the control of cerebral somatostatin (SS), we studied the effect of endogenous VIP blockade on immunoreactive SS (IR-SS) accumulation by fetal rat cerebral cortical and hypothalamic cells in culture. Cells were cultured in minimum essential medium (MEM) with 10% fetal calf serum and 10% horse serum. After 7-10 days 'in vitro' media were replaced with MEMs without sera containing anti-VIP immunoglobulins G (IgG) for 1, 3, 6, 24 or 48 h. Controls received the same amount of IgG from normal rabbit serum (NRS). In another group of experiments, cells were incubated with VIP (10(-11) M to 10(-7) M) for 1, 3, 6 or 24 h. Exposure to anti-VIP IgG resulted in a decreased accumulation of IR-SS in both cerebral cortical and hypothalamic cells, whereas the addition of VIP caused a dose-dependent increase in total IR-SS, these effects being evident after 3 h incubation. The stimulatory action VIP on IR-SS was up to 129%, this being decreased to 86% by the addition of anti-VIP to plates containing 10(-7) M VIP. Patterns of IR-SS accumulation throughout prolonged incubation periods were qualitatively similar (in both cerebrocortical and hypothalamic cells) in the presence or absence of anti-VIP IgG. However, in plates containing anti-VIP, the total amount of IR-SS was lower than in the control groups (IgG from NRS). These findings demonstrate that, at this time of brain development, somatostatinergic neurons may be under the physiological regulation of locally produced VIP.     

Effect of Complement and Viral Filtration on the Neutralization of Respiratory Syncytial Virus

The addition of 10 hemolytic units of guinea pig complement has been shown to enhance the neutralizing capacity of respiratory syncytial (RS) immune sera produced in guinea pigs and ferrets. This same immune sera, when tested without complement, had little or no neutralizing capacity. The addition of complement to RS immune horse serum did not significantly increase its neutralizing capacity. Immune horse serum effectively neutralized RS virus without complement. Other studies indicated that a 50% tissue culture infective dose of between 30 and 100 should be used in RS serum neutralization tests and that incubation should be for 90 to 105 min at room temperature. The neutralizing capacity of guinea pig immune serum was not increased by the use of filtered virus. The rate of virus neutralization, however, was increased with the addition of 10 hemolytic units of complement. The neutralizing capacity of RS immune horse serum was much greater for filtered than for unfiltered RS virus. The addition of complement increased the rate of virus neutralization but did not increase the neutralizing capacity of the horse immune serum.     

The effects of antibodies and complement in macrophage-mediated cytotoxicity on metacercariae of the lung fluke, Paragonimus westermani

Paragonimus westermani is a tissue migrating parasite in the early stage until arriving at lung, and most of the parasites spend their life spans there. Considerable immune responses including activation of macrophages are taken place during the residence of parasites in the host. However, concerning the immunologic defense mechanisms of the host against this parasite, only a few document is available so far. In this study, the cytotoxic effect of peritoneal macrophages under the presence of antibody and/or complement against metacercariae of P. westermani was investigated in vitro. Metacercariae were collected from the crayfish, Cambaroides similis and hatched out in Tyrode solution (pH 7.4). Plastic adherent cells from normal or infected rat (Wistar) peritoneal exudates were used as experimental macrophages. Polyclonal antibodies were obtained from infected rats and a cat. Cat IgG was fractioned with ion exchange chromatography. Fresh rabbit complement was used according to experimental scheme. Various combinations of peritoneal macrophages, normal or infected rat serum, complement and cat IgG were incubated at 36 degrees C in 5% CO2 incubator for 6, 14, 24 and 48 hours. The results obtained were as follows: 1. P. westermani infection activated peritoneal macrophages non-specifically and this activation induced increases of cell adherence and cytotoxicity on metacercariae. 2. In the presence of infected rat serum the antibody-dependent cell-mediated cytotoxicity of peritoneal macrophages on metacercariae was significantly increased and showed a peak at 6-hour incubation. But the cytotoxic effect was markedly reduced after inactivation of complement and heat-labile IgE antibody by the heating of infected serum at 56 degrees C for 30 minutes. 3. The highest cytotoxic effect (100%) of concomitant incubation with IgG and complement showed 24 hours after incubation, although cell adherence was relatively low at 6-hour incubation and 0% at 24-hour incubation. 4. Coordinative functions of complement with serum and IgG were effective in cell adherence and in cytotoxicity, but it is not clear the independent role of complement on the macrophage-mediated cytotoxicity in this study. With these results it is assumed that P. westermani infection can induce the non-specific activation of peritoneal macrophages, and serum antibodies including IgE antibody might enhance the cytotoxicity by macrophages.     

Cytotoxins - cause of serum activity as complement in the lymphocytotoxic test

The Ferrone's hypothesis elucidating the effect of rabbit complement in HLA typing using the cytotoxic test, due to the presence of xenocytotoxins to human lymphocytes in the rabbit serum was corroborated by the following experiments: 1. In human serum active as complement in HLA typing non-HLA lymphocytotoxins with optimum activity at 20 degree C were shown. In the guinea pig complement ineffective in HLA typing no cytotoxin to human lymphocytes were found. 2. In the rabbit complement cytotoxins to peripheral human lymphocytes were found when the incubation period of the cytotoxic test was prolonged to 3-4 h. 3. During the cytotoxic test on a model of rabbit immune sera - rabbit lymphocytes human complement showed a substantially higher activity than the rabbit complement. In the human complement xenocytotoxins to rabbit lymphocytes were demonstrated.     

Immune immobilization of Treponema pallidum: antibody and complement interactions revisited

The Treponema pallidum immobilization test was designed for serodiagnosis of syphilis and is dependent upon specific antibody and a heat labile component of normal serum. Investigators have shown the component to be dependent upon divalent cations and it is presumed to be complement. Experiments were performed to reevaluate the interactions of antibody and complement and the mechanism of immobilization. The loss of treponemal motility was correlated to the loss of complement activity in the reaction mixture. When motility of treponemes incubated with immune serum IgG and complement had dropped to 50% (3.4 h), 72% of the available complement had been consumed. At the same time, treponemes incubated with normal serum IgG and complement were 82% motile and only 51% of the complement had been consumed. C6 deficient rabbit serum and C4 deficient guinea pig serum were used in conjunction with immune serum IgG to determine which components of the complement cascade were necessary for immobilization. Treponemes were not immobilized by either sera. Results suggest that the heat labile factor in normal sera is complement, that both early and late components of the complement cascade are necessary, and that the reaction proceeds via the classical complement pathway. Although T. pallidum is susceptible to the actions of antibody and complement, the organisms must interact with these components for at least 2 h before immobilization will result.     

Subclasses of rat IgG active in the killing of schistosomula of Schistosoma mansoni in vitro and in vivo

Schistosomula of Schistosoma mansoni are known to be killed in vitro by complement and IgG (lethal antibody). To investigate whether this mechanism reflects the in vivo situation, we isolated IgG subclasses from sera of infected rats and assayed their ability to promote the complement-mediated killing of schistosomula in vitro as well as to protect normal recipients from a challenge infection. We found that a serum fraction containing only IgG2a + IgG2b has lethal activity to schistosomula in vitro, whereas a fraction containing only IgG1 + IgG2c fails to kill schistosomula in the presence of complement. The assay of protective activity has shown that the same fraction containing the lethal activity (IgG2a + IgG2b) was able to reduce the number of schistosomula recovered from lungs. These results provide evidence of the participation of IgG2a and/or IgG2b, but not IgG1 or IgG2c, in protective immunity to S. mansoni in rats, possibly through a complement-mediated mechanism.     

Lytic rabbit IgG for tissue culture trypomastigotes of Trypanosoma cruzi alters the extent and form of complement deposition

Infective and vertebrate stages of Trypanosoma cruzi are resistant to lysis by the alternative pathway of complement. To further elucidate the mechanism of complement evasion and to study how some immune sera render the infective stage sensitive to lysis, we compared the interaction of complement components C3 and C9 with the surface of complement susceptible, vector stage epimastigotes and vertebrate stage trypomastigotes of T. cruzi. Our studies showed that, upon incubation in human serum, complement resistant tissue culture trypomastigotes (TCT) bound five- to eightfold less C3 or C9 than complement sensitive epimastigotes (Epi). C3 bound to Epi is mainly in the hemolytically active C3b form, while TCT bear predominantly the hemolytically inactive iC3b fragment, which cannot participate in C5 convertase formation or lead to deposition of the lytic C5b-9 complex. Three- to sixfold more C3 and two- to threefold more C9 were deposited on TCT when lytic rabbit immune IgG with broad specificity was used to sensitize the parasites, and nearly one-half of bound C3 was present as C3b. In contrast, a comparison of three different sources of IgG from immune human serum showed a less clear correlation between the titer or specificity of anti-T. cruzi antibody, enhancement of C3 or C9 deposition, change in the form of bound C3, or killing. These results show that lytic rabbit IgG for T. cruzi changes the form and amount of bound complement components in anticipated fashion, but that human immune IgG does not give predictable changes in the extent or form of C3 or C9 deposition.     

Involvement of host Fc receptors in antibody-mediated cutaneous basophil hypersensitivity reactions.

Cutaneous basophil hypersensitivity (CBH) reactions are heterogeneous delayed time course basophil-rich responses that can be mediated by either T cells, B cells, or serum antibodies. The current study examined the mechanism by which antibodies mediate CBH in guinea pigs. Fc competition experiments were constructed by passively transferring mixtures of anti-KLH serum and normal heterologous gamma-globulins. It was found that rabbit IgG and its isolated and purified Fc fragment [but not the (Fab')2 fragment] inhibited the ability of guinea pig immune serum to transfer CBH. Concurrent inhibition of transferred KLH-specific CBH and systemic passive cutaneous anaphylaxis (PCA) reactions by rabbit IgG or its Fc fragment, and not by sheep or bovine gamma-globulins, indicated that Fc receptors on cutaneous mast cells were probably involved in both CBH and PCA. It was also found that the basophil aspect of delayed cutaneous responses elicited by PHA was inhibited by Fc competition maneuvers. This could mean that some forms of apparently T cell-mediated CBH may be T cell dependent, but via secretion of molecules that bind to Fc receptors, as seems required in antibody-mediated CBH.     

Effect of antilymphocyte serum on animals experimentally infected with Histoplasma capsulatum or Cryptococcus neoformans

The anti-mouse and anti-guinea pig antilymphocyte sera (ALS) prepared for this study were shown to contain cytoxic and leucoagglutinating antibodies, and were capable of producing severe lymphopenia in these animals. Guinea pigs treated weekly with ALS were more susceptible to development of fatal infection when inoculated with Histoplasma capsulatum. No fatalities occurred in guinea pigs infected with equal doses of H. capsulatum but treated with normal rabbit serum (NRS) or saline. The time necessary to reach 50% fatality in mice infected with Cryptococcus neoformans was greatly reduced by pretreatment with ALS in comparison with infected controls treated with NRS or saline. When low dosages were used (0.1 ld(50)), the effect was even more pronounced. Spleen homogenates from mice infected with equal dosages of H. capsulatum and treated with ALS or NRS were cultured. More than 150 times as many organisms were present in the spleens of the ALS-treated group. Similar results were obtained from culturing the lungs and liver. Delayed hypersensitive skin reactions were radically decreased or abrogated in H. capsulatum-infected guinea pigs inoculated intraperitoneally with ALS 12 hr before skin testing with histoplasmin. When ALS was given weekly, the influence on skin reactivity was less notable. Given intradermally, ALS was shown to inhibit the delayed reaction to histoplasmin within a radius of 40 mm.     

Antibodies against epididymal glycoproteins block fertilizing ability in rat

Antiserum against rat androgen-dependent secretory epididymal protein DE (raised in rabbit) was added to suspensions of rat spermatozoa from the cauda epididymidis which were used for artificial insemination. While control spermatozoa fertilized 41.6% of oocytes, those exposed to antiserum to protein DE fertilized only 6.6% (P less than 0.01). An equal amount of normal rabbit serum (NRS) did not cause inhibition (33.1%). To study the entry of antibodies into the epididymis, caudal tubules were cultured for 24 h and the fertility of the contained spermatozoa was assessed by artificial insemination. Culture in Medium 199 alone or with NRS resulted in spermatozoa which fertilized 52% of oocytes while the presence of antiserum to protein DE in the culture medium yielded spermatozoa which fertilized only 16.6% of oocytes (P less than 0.01). These results suggest (1) that the epididymal protein DE might be part of a sperm structure involved in the fertilization process, and (2) that, at least under the present culture conditions, immunoglobulins penetrate the epididymal epithelium in sufficient numbers to reduce fertility significantly.     

Taenia crassiceps: effect of normal and immune serum on metacestodes in vitro.

The effect of normal and immune serum on Taenia crassiceps larvae in vitro was assessed by Evans blue dye uptake and electron microscopy. Normal guinea pig, rabbit, goat, and fetal calf serum did not have any significant detrimental effects upon the larvae after 7 days of culture in vitro. Culture for 7 days in normal mouse serum resulted in some loss of tegumental microtriches but the tegument itself remained intact. Culture in hyperimmune rabbit serum resulted in complete loss of the tegument and disruption of subtegumental structures within 48 hr. The effects of immune mouse serum in vitro closely paralleled those previously seen during early immune damage in vivo. Immune serum taken 2 to 4 weeks after secondary intraperitoneal infection with T. crassiceps metacestodes caused loss of the larval tegument and degeneration of the subtegumental tissues after 7 days in culture, whereas immune mouse serum taken 6 weeks after secondary infection caused only minor ultrastructural changes and appeared to be less toxic to larvae than normal mouse serum. Although complement appeared to increase the number and severity of the tegumental lesions, the presence of heat-labile components of complement was not essential for mediation of tegumental damage by immune mouse serum.     

Production of anti-rat islet cell surface antibody in guinea pigs

Anti-rat islet serum was prepared in guinea pigs by multiple subcutaneous inoculations of rat islets homogenates emulsified in complete Freund's adjuvant (CFA). The anti-rat islet serum was cytotoxic against rat spleen cells in the presence of complement and the nonspecific antibodies were observed with homogenates of rat livers and spleens. After absorption, the serum lost the cytotoxicity against the rat spleen cells yet showed specific cytotoxicity against the rat islet cells. The binding capacity of anti-rat islet antibody was determined by the indirect immunofluorescence test using FITC conjugated rabbit anti-guinea pig IgG serum. As the guinea pig anti-rat islet serum contained anti-insulin antibody, the role of this antibody in this cytotoxic activity and surface immunofluorescence was studied. However, the anti-insulin antibody used as the control showed neither cytotoxicity nor surface immunofluorescence. After neutralizing the anti-insulin antibody in the antiserum with insulin, the serum remained cytotoxic to the rat islet cells and a surface immunofluorescence appeared. These data show that specific anti-rat islet cell surface antibody can be produced in guinea pigs by multiple inoculations of rat islets homogenates with CFA.     

Estimation and Identification of Streptococcus dysgalactiae Protective Antibody in Sera of Rabbits and Cattle

Rabbit and cow anti-Streptococcus dysgalactiae sera were tested by bacterial agglutination, complement fixation, hemagglutination, and immunodiffusion for the presence of antibody. The results of these tests were compared with mouse-protection studies on the same serum to estimate which in vitro test would best reflect the in vivo protective capacity of serum. Identification of the antibody constituents responsible for the mouse protection, hemagglutination, and complement fixation titers were established by reacting whole and diluted antisera with mercaptoethanol before and after testing. Results indicate that the complement fixation test may be a more accurate indicator of IgG protective bovine and rabbit antibody, whereas the hemagglutination test may more readily reflect a wider range of protective antibody levels and IgM. The complement fixation test showed some shared responses to IgG and IgM in both the rabbit and cow, whereas the IgM components seemed to be the predominant factor influencing hemagglutination titers in the rabbit and more so in the bovine. Mouse protection tests with mercaptoethanol-treated cow and rabbit sera indicate that the protective capacity of these antisera is shared between IgM and IgG components.