Classical and alternate complement pathway activities in paired dairy cow-newborn calf sera

Hemolytic assays were used to compare alternate and classical C pathway activities in sera obtained from clinically normal newborn dairy calves and their mothers at the time of delivery. Mean alternate and classical CH50 concentrations in sera from newborn calves were both significantly lower than in their dams (P less than 0.001). The titer of alternate C pathway activity, expressed as CH50 units/ml, in sera from 17 calves was 12.9 +/- 5.5, whereas for the cows it was 25.8 +/- 6.2. The ratio of cow: calf serum alternate CH50 titers averaged 2.25 +/- 0.80 and ranged from 0.88 to 4.14. Classical CH50 titers were 78.0 +/- 42.7 units/ml in calf sera and 246.0 +/- 44.5 in cow sera. The ratio of cow: calf serum classical CH50 titers averaged 3.71 +/- 1.49 and ranged from 1.19 to 6.87. The wide range of values, noted for both the alternate and classical C pathways, within maternal and neonatal groups was assumed to reflect the biologic variability of complement levels in bovine serum. The possible relationships between deficient levels of alternate and classical CH50 activity in newborn calves and their susceptibility to infections is discussed.     

A study of the sensitivity of erythrocytes to lysis by heterologous sera via the alternative complement pathway

In order to get insight in the distribution of alternative complement pathway activities as detected by lysis of xenogeneic erythrocytes in the presence of magnesium and ethyleneglycol-bis-(2-aminoethyl)-tetra-acetic acid (EGTA) over the species, the 156 heterologous combinations of erythrocytes and sera out of thirteen animal species were tested. An order could be noticed in the species with respect to serum complement activity tending to negative correlation with the sensitivity of the corresponding erythrocytes to lysis by heterologous sera. So far, the most sensitive erythrocyte for each individual serum must be considered to be the target cell of choice for developing assays for alternative complement pathway activity in the serum involved. In this series of animals only for rabbit serum no sensitive target cell was found. The order observed, in connection with the failing lysis of erythrocytes by homologous sera, suggests further that in restriction of heterologous hemolysis in general one erythrocyte-associated, species-nonspecific regulatory principle may be involved, whereas in homologous restriction, most probably, also species-specific factors play a role.     

Hemolytic assay for goat (caprine) and swine (porcine) complement

Optimal conditions for assaying hemolytic complement of goat (caprine) and swine (porcine) sera were determined. Effects of the following were tested: pH, ionic strength, calcium and magnesium ion concentrations, time and temperature of incubation, and ethylenediamine tetracetate concentration. Guinea pig erythrocytes sensitized with goat or cattle antibodies were the most sensitive target cells for goat complement. Sheep and cattle erythrocytes sensitized with rabbit hemolysin were the best target cells for swine complement. Barbital buffer, pH 7.3, ionic strength of 90 nmM relative salt concentration, containing 0.5 mM CaCl2 and 1 mM MgCl2 was the best for swine complement assay. Goat complement lysed best in a barbital buffer, pH 8, ionic strength of 90 to 120 mM of relative salt concentration, in presence of 0.5 mM CaCl2 and 1 mM MgCl2. The optimal incubation temperature was 37 degrees C for both complements. The complement dependent lysis required 75 minutes to reach its maximum. Ethylenediamine tetracetate in 4 mM concentration completely inhibited lysis by both species complements. The CH50 for goat sera varied between 18 and 75 per ml, in swine sera between 75 and 210 per ml.     

Classical and alternative pathway haemolytic activities of ovine complement: variations with age and sex

The classical (CH50) and alternative (ACH50) pathway haemolytic activities of sheep complement were measured with microtechniques. Storage of blood at room temperature (instead of 4 degrees C) before centrifugation and usage of sera stored at -70 degrees C were compatible with complement titration. The effects of age and sex were tested in 303 sera obtained from animals aged between 2 weeks and 3.5 years old. ACH50 titres were low during the first 1.5 months of life then increased to reach the level found in adults at the age of 3 months. Conversely, CH50 titres were very high in suckling lambs, decreased up until the age of 3 months and then increased to reach the adult level at 1 year old. In lambs, the haemolytic complement activity was significantly higher in females than in males.     

Bovine IgM: does it fix guinea pig complement in the absence of bovine complement components?

Purified bovine isotypes IgM, IgG1, IgG2, and IgA (secretory), affinity purified with Brucella abortus, were tested in a complement fixation test (CFT) for their ability to activate guinea pig complement directly or in the presence of 'normal' bovine serum. Only IgG1 fixed guinea pig complement in the direct test and approximately 250 ng of antibody was required to activate 50% of 3 CH50 units with a standard amount of antigen. Addition of 'normal' bovine serum as an additional source of complement resulted in activation of guinea pig complement by IgM, IgG2 and secretory IgA at levels of approximately 1200, 700 and 2250 ng, respectively for 50% of 3 CH50 units. Addition of 'normal' bovine serum did not enhance complement activation by bovine IgG1.     

Serodiagnosis of canine herpesvirus infection--development of an enzyme-linked immunosorbent assay and its comparison with two improved methods of serum neutralization test

An enzyme-linked immunosorbent assay (ELISA) for the diagnosis of canine herpesvirus (CHV) infection using antigen prepared by solubilizing infected cells was developed. The ELISA and two improved methods of serum neutralization test, the microplate serum neutralization test (MSNT) with complement and the 50% plaque reduction (PR) assay with complement, were compared for the results of antibody detection from a total of 557 field canine sera. Of 529 sample sera that were negative in the MSNT with complement, 119 were ELISA positive, and this result together with time course of serum antibody detection in a dog experimentally infected with CHV strongly suggested that the MSNT with complement is less sensitive for the detection of antibody in CHV infected dogs, especially those in early stages of infection. A correlation was found between the titers measured by the ELISA and 50% PR assay with complement, however, for field use, the ELISA is recommended as a highly sensitive test method of serodiagnosis of CHV infection adequate for dealing with a large number of samples with less demand on time and effort.     

Antibody dependent haemolysin, complement and opsonin in sera of a major carp, Cirrhina mrigala and catfish, Clarias batrachus and Heteropneustes fossilis

The present communication is a continuation of our earlier study on the natural serum haemagglutinin/lectins of Cirrhina mrigala, Clarias batrachus and Heteropneustes fossilis. Sera of Cirrhina mrigala, belonging to the major carp family, could not only agglutinate heterologous rabbit erythrocytes, but also lyse them spontaneously. This lysis of rabbit RBC by Cirrhina mrigala sera was calcium ion dependent and heat sensitive, indicating thereby that the haemolysis was mediated by the fish serum complement system via the classical pathway. Quantification of CH50 and APCH50 levels in the sera of Clarias batrachus and Heteropneustes fossilis as well as in the sera of amphibia, aves and mammals showed that lower vertebrates predominantly possessed an alternative pathway of the complement system, while on the other hand, in the higher vertebrates the major pathway of complement activation was classical. Furthermore sera of Clarias batrachus and Heteropneustes fossilis had opsonins, which could stimulate heterologous rat peritoneal macrophages to engulf Staphylococcus aureus with the production of superoxide anion. From this study we concluded that fishes have been armed with various powerful natural humoral defense systems for their protection against environmental pathogens.     

A hemolytic assay for the measurement of equine complement

A hemolytic assay was developed for the measurement of functional equine complement activity. The assay utilizes antibody sensitized chicken erythrocytes as the target cell and was specific for classical pathway (antibody dependent) complement activity. The assay was found to be reproducible and more sensitive than previous reports using other species of target cells. Total serum complement (CH50) values were determined for five mares and their foals and followed over a period of 3 months.     

Hemolytic complement levels of neonatal calves delivered from protein-energy malnourished dams and subjected to cold stress

Beef cows were placed on protein-deficient and/or energy deficient rations for the last 150 days of pregnancy. After birth their calves were placed on 1 or 21 C environmental chambers for 3 days, and sera were collected for determination of complement (C) levels. At birth, the mean complement hemolytic (CH50) titer of all calves was 46.0 +/- 1.7 units, but the titer rapidly dropped (P < 0.01) to 31.6 +/- 1.2 by 12 hours after birth. Levels of C activity then began to rise and reached a mean titer of 76.3 +/- 3.0 by 3 days of age. A quadratic curve of predicted CH50 values was constructed from the data. Differences between principal and control groups of calves were not detected. These results suggest that maternal protein-calorie deprivation and limited cold stresses have little effect on levels of C activity in the bovine neonate. Possible explanations for the decrease in CH50 levels after birth are discussed.     

Failure of bovine complement levels measured by radial hemolysis to correlate with tube titration

Hemolytic complement levels in 30 duplicate samples of normal bovine sera were determined by a tube titration method (CH50) and by a radial hemolysis in gel assay. Significant correlation was not observed between the values obtained by the 2 tests. This lack of correlation could be a result of considerable error observed in values obtained for duplicate samples in the CH50 method or to the relative insensitivity of the hemolysis in gel test. The hemolytic reaction in gel was found to depend on Mg2+, thus raising questions (i) about its validity in determining total hemolytic complement or (ii) about bovine C1q depending on Mg2+ rather than Ca2+ for binding to immune complexes.     

The anticomplementary effect of animal sera--a contribution to the precise definition of the complement-binding reaction

Photometric procedures (standardised complement fixation reaction) were used to determine the anticomplementary action of animal sera. Multiplication factors, K, of 3.0 to 5.0, related to human sera, were recorded and were found to be capable of reducing or offsetting anticomplementary serum action in CFR in terms of a definitive complement excess. Anticomplementary action of animal sera, for all practical purposes, does no longer apply to dilution ratios of 1:16. The complement excess necessary for titres of 1:4 and 1:8 should be precisely defined and can then be calculated in constant measures. Hence, titres of 1:8 may be rendered accessible to safe assessment. Titres of 1:4 will continue to be problematic. The following order of animal sera was defined in comparison to human sera: cattle, pigeon, duck, sheep, cat, turkey, swine, coypu, hen, dog.     

Antibodies to Aujeszky's disease virus in pigs immunized with purified virus glycoproteins

Antibodies to Aujeszky's disease virus (ADV) glycoproteins gII, gIII, and gp50 were compared using four in vitro tests. Antibodies generated by vaccination with a modified-live vaccine (MLV) were also compared. The serological assays employed were: serum neutralization test (SNT), complement facilitated serum neutralization test (C'SNT), complement-mediated cytolysis and antibody dependent cellular cytotoxicity (ADCC). Pigs were immunized with single glycoproteins twice 14 days apart, or once with the modified-live vaccine. Fourteen days after the second immunization, sera were collected. Virus neutralizing activity (SNT) was demonstrated in the sera from all pigs immunized with gp50 and in one out of three immunized with gIII. Sera from the MLV group all had neutralization titers higher than animals immunized with single glycoproteins. Addition of guinea pig complement to the serum neutralization test (i.e., C'SNT) produced an enhancement of antibody titers in all groups except the pigs immunized with gIII. The complement-mediated cytolysis test rendered antibody titers similar in magnitude for all pigs immunized with single glycoproteins, but slightly lower than values for MLV vaccinated pigs. ADCC activity was clearly displayed in sera from pigs immunized with gIII or vaccinated with MLV, whereas sera from pigs immunized with gII or gp50 had a minimal response. The results indicate that the relative efficiency of antibodies against ADV glycoproteins in protection should be considered for selecting or producing gene-deleted strains for use in vaccine production.     

Influence of major histocompatibility genes on serum hemolytic complement activity in miniature swine

Total serum hemolytic complement (CH50) activity was determined for 3 semi-inbred strains of miniature swine (SLAa, SLAc, and SLAd) and 1 recombinant strain SLAg (ABCcDd), each homozygous for a distinct major histocompatibility complex haplotype. Initial determination was made at 8 weeks of age, prior to standardized immunization, the second at age 12 weeks, after immunization. Analysis of variance was by least-squares method, using a linear model on data from 33 litters by 14 sires and 16 dams. Analysis of variance indicated that the combined effects of haplotype, sire, dam, litter, and gender accounted for 47.63% of the total variation in preimmunization CH50 values. Dam (P less than or equal to 0.06) and litter (P less than or equal to 0.03) significantly influenced preimmunization complement activity. Although swine leukocyte antigen (SLA) haplotype was not significant in the model, least-squares mean comparisons between haplotypes suggested that ac, dg, and gg pigs tended to have comparatively low preimmunization CH50 values. The model did not account for significant variability in postimmunization CH50 values, but least-squares means indicated that dd, dg, and gg haplotypes tended to have lower values than did other haplotypes tested. Mean CH50 units for 8- and 12-week-old pigs were 41.32 +/- 20.49 and 59.50 +/- 54.35, respectively. There was a significant difference (P less than or equal to 0.001) in CH50 activity between 8- and 12-week-old pigs associated with immunization, because CH50 of nonimmunized controls did not differ at 8 and 12 weeks.     

Antibodies against a tumour-associated antigen in an AKR lymphoma conditioned to grow in BALB/c mice

An experimental model was used in which AKR lymphoma cells (L15) were conditioned to grow in BALB/c mice leading to tumour-bearing (progressor) and tumour-rejecting (regressor) animals. The behaviour of antibodies present in the sera of these animals was studied using as antigen L15 cells or a soluble tumour-associated antigen TEs. Both sera showed similar IIF and haemagglutinating activity. However differences were observed for the complement cytotoxicity assay. Regressor serum as well as a rabbit anti-tumour-associated antigen serum were strongly cytotoxic for the AKR lymphoma cells while progressor serum showed markedly lower activity. Specific antibodies against the tumour-associated antigen were purified. In both sera they were located mainly in IgG1 but also in IgG2. The purified antibodies agglutinated specifically sensitized sheep erythrocytes and reacted by indirect immunofluorescence with L15 cells but not with AKR thymocytes. It is suggested that two qualitatively different humoral immune responses are involved in the mechanisms leading to tumour enhancement or rejection.     

Further evidence that bovine IgM does not fix guinea pig complement

In a study of sera from cattle vaccinated with 3 X 10(10) cfu of Brucella abortus strain 19, it was found that IgG1 antibody measured by an indirect ELISA was the only isotype to correlate with standard complement fixing antibody titers using heated serum samples and guinea pig serum as a source of complement. A supplement of normal unheated bovine serum resulted in IgM fixing guinea pig complement, giving data similar to those obtained with unheated serum in the complement fixation test.     

The effects of five different glycans on innate immune responses by phagocytes of hybrid tilapia and Japanese eels Anguilla japonica

The aim of this study was to evaluate the immune responses in hybrid tilapia (Nile tilapia Oreochromis niloticus x Mozambique tilapia O. mossambicus) and Japanese eels Anguilla japonica after treatment with five glycans: barley, krestin, MacroGard, scleroglucan, and zymosan. The effects of the glycans on the innate immune responses of the fish were investigated using the phagocytic index (PI), lysozyme activity, complement opsonization, and activation assay. The results of the lysozyme assay demonstrated that the lysozyme activities increased after treatment with glycans. Moreover, based on the PI, treatment with each of the five glycans resulted in increased phagocytic activities in anterior kidney and peripheral blood phagocytes in both tilapia and Japanese eels. The opsonic effect of complement on phagocytosis in tilapia and Japanese eels were investigated using baker's yeast, which served as the activator in the classical complement pathway (CCP) and in the alternative complement pathway (ACP). Tilapia and Japanese eel sera that were treated with glycans greatly enhanced phagocytosis. The classical pathway--hemolytic complement titer (CH50) of Japanese eels treated with glycans was slightly increased in vitro and in vivo. While glycan treatment enhanced the CCP of both species in vitro and in vivo, the alternative pathway-hemolytic complement titer (ACH50) was only increased in vitro and in vivo in glycan-treated tilapia. Thus, it follows that the ACP must have been activated in tilapia treated with glycans. However, in Japanese eels, the ACH50 of the ACP activation assay was undetected in vitro or in vivo due to possible unknown factors in the Japanese eel serum that caused lysis of the rabbit red blood cells. Our study investigated the effects of glycans used to enhance phagocytosis and activate both of the complement pathways involved in stimulating the innate immune responses of Japanese eels and tilapia.     

Effect of the caprine variant of Mycoplasma mycoides subsp mycoides on endothelium, monocytes, and complement of guinea pig, calf, sheep, and goat serum

The caprine variant of Mycoplasma mycoides subsp mycoides causes septicemia with coagulopathy in goats. Pathogenetic mechanisms that might explain the coagulopathy, the ability of the Mycoplasma to persist in the blood, and its specificity for goats were studied. Severe endothelial damage was seen by electron microscopy of goat aorta tissue exposed in vitro to 10(7) colony-forming units of mycoplasmas. The Mycoplasma did not damage 51Cr-labeled adherent cells from peripheral blood of goats. The hemolytic complement titer was reduced by 94%, 50%, 50%, and 25% in guinea pig, calf, sheep, and goat serum, respectively, 30 minutes after treatment with 8 X 10(9) colony-forming units of the Mycoplasma. Freshly prepared serum from these animal species killed the Mycoplasma. Heat-inactivated serum was not mycoplasmacidal. Complement from these 4 animal species was activated by the Mycoplasma through the classical pathway, because ethyleneglycoltetraacetic acid precipitation of serum Ca2+ inhibited activation. Proof that the classical pathway was functional in goats was not conclusive because Ca2+ supplementation of ethyleneglycoltetraacetic acid-treated serum did not restore complement activity. Endothelial damage and complement activation may explain the coagulopathy. The function that complement activation may have in the inflammatory response of this disease is not known. Difference in susceptibility of calves, sheep, and goats to M mycoides septicemia cannot be explained by species variation in complement mycoplasmacidal activity.     

Studies on activation and levels of haemolytic complement of buffalo (Bubalus bubalis). II. Alternate complement pathway activity in serum.

Buffalo serum caused lysis of unsensitized red blood cells (RBC) of sheep, goat, rabbit and guineapig. There was minimal lysis of cattle RBC, and homologous RBC were resistant. Lysis of sheep and goat RBC was the result of natural antibodies as adsorption with respective RBC and addition of 8 mmol ethylene glycolbistetraacetate (EGTA) in diluent completely abrogated the haemolytic activity. The lysis of guinea-pig and rabbit RBC was only partially decreased by these treatments, indicating the presence of alternate complement pathway (ACP) activity in buffalo serum. The guinea-pig RBC were the most sensitive to lysis, and 50% CH titre units above 40 ml⁻¹ of serum were obtained. The haemolytic activity of buffalo C for unsensitized guinea-pig RBC was reduced from 47 CH₅₀ units to an undetectable level by heating at 50°C for 20 min and at 56°C for 4 min. Similarly, treatment with zymosan also inhibited this haemolytic activity. Maximum activation of buffalo ACP occurred in the presence of 4 mmol Mg²⁺ in the diluent.

Using standardized conditions, ACP activity was determined in sera of 98 healthy buffaloes of different age groups from 1 month to 12 years. Even young calves less then three months of age showed considerable ACP activity (45.60±1.21 CH₅₀ units ml⁻¹) which increased with age. The peak mean values of 79.79±1.45 CH₅₀ units was recorded in 2 to 4-year-old animals. However, in all the 11 animals above 4 years of age, the haemolytic activity was greatly reduced and was even less than that in 1 to 3-month-old buffalo calves. Haemolytic activity did not vary between the sexes.     

A comparative study of complement activation

Antisera to sheep erythrocytes (E) were raised in cattle, rabbits, mice, hamsters, guinea-pigs, ferrets, badgers, hedgehogs and fowls. Cross activation of total haemolytic complement (THCA) examined all combinations of sensitized sheep E and normal sera (including human); kinetic assays examined the lysis of E sensitized with rabbit antibodies. From the same species, all combinations of normal serum and xenogeneic E were used to measure total alternative pathway activity (TAPA); TAPA was also activated by rabbit and sheep E in titrations and in agarose gels, and examined kinetically against rabbit E. Ox, rabbit and fowl sera were low in THCA, guinea-pig complement was universally active, while human complement showed marked selectivity; ferret, badger and hedgehog sera were activated to high titres but probably via the alternative pathway. In studies of TAPA an inverse relationship existed between serum complement activities and the activating abilities of E from the same species. The most efficient activators of alternative pathway were E from rabbits and laboratory rodents, while the sera with broadest response were badger, ferret and fowl. Kinetic studies of TAPA showed that initiation of lysis and subsequent completion of lysis could occur with different efficiencies, suggesting these events reflected separate events in complement activation.     

The role of specific immunoglobulins in antibody-dependent cell-mediated cytotoxicity assays during Babesia bovis infection

A stabilate prepared from Babesia bovis-infected Boophilus microplus ticks was used to infect intact adult cattle. Whole sera and immunoglobulin fractions from representative sera were tested by complement fixation (CF), indirect fluorescent antibody (IFA), and antibody-dependent cell-mediated cytotoxicity (ADCC) assays. The last test utilized 51Cr-labeled chicken erythrocytes coated with Babesia bovis antigen as targets. Mononuclear cell preparations, obtained from peripheral blood of normal donors and consisting of lymphocytes with 2--6% large monocytes, were used as the source of effector cells. Antibody activity was detected by all tests between 14 and 16 days following infection. Specific IgM and IgG1 were reactive in both CF and IFA tests, although the development of high titers was attributable to IgG, alone. The ADCC activity was restricted to IgG1 fractions and was greater in those sera or fractions with greater CF activity. No activity was demonstrated in IgG2 fractions by any test used.