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.     

Demonstration of Alternative and Classical Complement Pathway Activity in Colostrum from Buffalo (Bubalus bubalis)

Buffalo colostrum caused lysis of unsensitized red blood cells (RBC) from sheep, goats, rabbits and chickens. RBC from cattle and buffalo were resistant to lysis. That lysis was due to the presence of natural antibodies to these RBC was ruled out since there was no reduction in haemolytic titres even after adsorption with the respective RBC. The addition of EGTA to the diluent had no effect on the haemolytic activity. These findings indicate the presence of alternative complement pathway (ACP) activity in buffalo colostrum. The haemolytic activity of buffalo complement for unsensitized rabbit RBC was reduced to very low levels by heating at 50°C for 45 min. Treatment with zymosan also inhibited the haemolytic activity, while inulin had no effect. The maximum activity of ACP occurred in the presence of 4 mmol/L Mg²⁺ in the diluent. The range of ACP activities in colostrum from buffaloes varied from 4.06 to 8.48 CH₅₀ units/ml. Using a standard system for titrating the classical complement pathway and rabbit red blood cells sensitized with goat haemolysin, the range of complement activity in buffalo colostrum was 4.81–6.77 CH₅₀/ml.     

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.     

In vitro studies on feline panleucopaenia virus. Standardisation of haemagglutination-inhibition test for feline panleucopaenia virus antibody

Standardised procedure for obtaining reproducible haemagglutination-inhibition results for FPV antibody which correlate with serum-neutralization titres was described. Optimal conditions were found to be Alsevers anticoagulant, PBS/0.05% BSA (pH 6.8) as buffer, especially washed round bottom microplates, determination of maximally sensitive porcine erythrocytes, use of reproducible erythrocyte concentrations, inactivation of serum samples at 56 degrees C for 30 min and serum treatment with koalin pH 9.0. The concentration of erythrocyte used for estimation of haemagglutination units in H1 test should not differ from that used as indicator in the test. Predilution of serum beyond 1:4 associated with false results. Reproducible method for removing natural agglutinins in serum by adsorption with erythrocytes was described.     

Haemagglutination patterns of the different variants of Escherichia coli K88 antigen with porcine, bovine, guinea pig, chicken, ovine and equine erythrocytes

Determination of the porcine adhesive phenotype was not achieved by haemagglutination (HA) of porcine erythrocytes, which in all cases were agglutinated by K88ab and K88ad, independent of the adhesive phenotype as determined by the brush border adhesion test. K88ac always gave negative HA results with porcine red cells. However, HA appeared to offer a method of differentiating between the K88 variants without monospecific antisera. K88ab agglutinated porcine, guinea pig and chicken erythrocytes; K88ac agglutinated only guinea pig red cells and K88ad produced haemagglutination with porcine and guinea pig erythrocytes.     

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.     

The use of a passive hemolysis system to evaluate the complement activities of six mammalian species

A passive hemolysis assay system was developed which permitted comparisons of the hemolytic activities of complement (C) from six species. This system employs a single antigen and an antiserum raised in one species. Thus, variations resulting from different target antigens and those inherent in using antibodies (of different affinities and isotypes) raised in a variety of species were minimized. Of the erythrocytes (E) examined, those from horses and guinea pigs were most susceptible to lysis, and either would be suitable, as a tentative choice, for measuring C activity of a previously unstudied species. Horse serum had the lowest C activity of any of the sera tested. It lysed certain cells only at high concentrations, and the hemolytic activity dropped off sharply with minimal dilution. The data presented in this paper could also be used for selecting target E for C studies using direct lysis with antibodies raised against the cells.     

Serologic response to Brucella abortus of cattle and guinea pigs experimentally inoculated with a Yersinia enterocolitica serotype from milk

Ten strains of Yersinia enterocolitica belonging to ten various serogroups isolated from raw milk were inoculated into groups of five guinea pigs and five calves. Y. enterocolitica serotype 0:16 was the only serotype tested that induced an antibody response to Brucella abortus in calves. No anti-Brucella response could be demonstrated serologically in guinea pigs. Activity of the anti-Y. enterocolitica 0:16 calf sera against B. abortus antigen was shown by the tube agglutination test, and by the complement fixation test. The early agglutinating antibody response was partly sensitive to reduction by 2-mercaptoethanol. This sensitivity decreased later in the response. This is the first report of anti-Brucella responses induced by a serotype of Y. enterocolitica other than 0:9; sera from a group of five calves inoculated with 0:9 were tested by the same serological techniques for comparison.     

猪圆环病毒2型免疫过氧化物酶单层细胞试验抗体检测试剂盒的研制及应用

本研究建立了一种免疫过氧化物酶单层细胞试验（Immunoperoxidase monolayer assay,IPMA）,用于猪圆环病毒2型血清抗体检测,通过对IPMA反应条件的优化,组装了诊断试剂盒。研究结果表明,用IPMA检测猪圆环病毒2型人工感染猪血清,于感染后3周抗体阳转,第3周～10周抗体阳性检出率为92.8%（52/56）,对照组猪血清抗体检测均为阴性（33/33）。试剂盒在-20℃稳定保存18个月与其他几种猪病毒参考血清无交叉反应,与用重组蛋白抗原建立的rcELISA符合率为89.2%。对来自黑龙江、吉林、河北、上海、内蒙古、云南、江西等地猪场健康成年猪血清480份和发病猪血清424份进行了检测,抗体检出率分别为91.7%和79.2%,表明我国猪群中猪圆环病毒2型污染相当严重。该试剂盒的研制为我国PCV2流行病学调查和疫苗免疫效果的评价提供了技术手段。     

Improving a Complement-fixation Test for Equine Herpesvirus Type-1 by Pretreating Sera with Potassium Periodate to Reduce Non-specific Hemolysis

Non-specific hemolysis has often been observed during complement-fixation (CF) tests for equine herpesvirus type-1 (EHV-1), even when the sera have virus-specific CF antibodies. This phenomenon has also been reported in CF tests for various infectious diseases of swine. We found that the sera from 22 of 85 field horses (25.9%) showed non-specific hemolysis during conventional CF testing for EHV-1. Because pretreatment of swine sera with potassium periodate (KIO₄) improves the CF test for swine influenza, we applied this method to horse sera. As we expected, horse sera treated with KIO₄ did not show non-specific hemolysis in the EHV-1 CF test, and precise determination of titers was achieved.     

Serological Diagnosis of Classical Swine Fever: A Comparison of a Modified Direct Complement Fixation Test with an Immunofluorescence Plaque Neutralization Test in the Diagnosis of Experimental Subclinical Infection

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.     

Development and characterization of mouse monoclonal antibodies to eight human complement components: Analysis of reactivity with orthologs of nine mammalian genera

To study complement function in mammalian leishmanioses, we developed mouse monoclonal antibodies to the human complement system components C1q, C4, factor D, factor H, factor B, properdin, C5 and C9. Antibody specificity was determined by indirect and capture ELISA and by Western blot. In flow cytometry analysis, seven antibodies recognized the cognate component on human serum-opsonized Leishmania promastigotes. Antibody reactivity was screened against promastigotes opsonized with sera of nine mammalian genera: pig, guinea pig, goat, rabbit, cat, dog, hamster, jird and rat. No antibody recognized jird epitopes on promastigotes. Anti-C4, -properdin, and -C5b reacted with the orthologous protein of all other mammals tested except cat (anti-properdin) and hamster (anti-C5b); anti-C9 only recognized the rabbit ortholog, and anti-C1q, -factor B and -factor H did not react with any of the nine orthologs. Such interspecies crossreactive antibodies can be valuable tools for analysis of mammalian complement function in infectious diseases.     

Tissue chamber model of acute inflammation in farm animal species

A tissue chamber model of acute inflammation for use in comparative studies in calves, sheep, goats and pigs has been established and validated. Tissue chambers were prepared from silicon rubber tubing, of inner diameter 12.7 mm, length 115 mm and volume 15 ml, with 10 holes, each of 6mm diameter, at each end. In each animal two or four chambers were inserted at subcutaneous sites. Six weeks after implantation an acute inflammatory reaction in a single cage was generated by the intracaveal injection of 0.5 ml of 1% carrageenan solution. Serial samples of exudate (injected chamber), transudate (non-injected chamber) and blood were collected for measurement of exudate and transudate leucocyte count, prostaglandin (PG)E(2) concentration in exudate and serum thromboxane (Tx)B(2) concentration. In addition, skin temperature changes over exudate and transudate chambers were recorded. In all four species, carrageenan induced an acute inflammatory response, indicated by increases to peak values followed by return towards baseline in skin temperature, leucocyte count and PGE(2) concentration. For each of these variables in calves, sheep and goats the increases were significantly greater for exudate than for transudate. The degree of intra-species variation in each variable was acceptable. Marked inter-species differences were recorded: skin temperature rise was greatest in calves and least in sheep and goats; exudate PGE(2) concentration was increased in the order sheep>goat>pig>calf; serum TxB(2) concentration was increased in the order calf>goat>sheep>pig and exudate leucocyte count was increased to a greater extent in the pig than in the three ruminant species. The model has advantages over some previously described tissue chamber models of inflammation and will be suitable for use in comparative studies of inflammatory mechanisms and the pharmacokinetics and pharmacodynamics of anti-inflammatory drugs.     

Defences of the bovine mammary gland against infection and prospects for their enhancement

The equine alternative complement pathway has been partially characterized and compared to the equine classical activation pathway. A dose-dependent lysis of RbRBC was observed with peak lytic values noted within 10 minutes at 37°C when rabbit red blood cells (RbRBC) were used as an alternative pathway activator. Sheep red blood cells (SRBC) sensitized with rabbit hemolysin or partially purified equine IgM antibodies were equally sensitive to lysis. Dilution of the commercial hemolysin by $\text{1}\text{5}$ reduced lysis from 90% to 38% in the presence of constant cell numbers. Hemolysis of SRBC peaked at 10 minutes and the majority of lysis occurred within 10 minutes. Dilution of equine sera by as little as $\text{1}\text{5}$ decreased hemolytic activity for SRBC to 21.5% from greater than 90% with undiluted sera. The alternative pathway protein, equine factor B, was tested using RbRBC and monitored by its differential susceptibility to heat treatment at 50°C. This treatment led to almost complete inactivation after a 15-minute incubation. An apparent heat-dependent decay of certain classical pathway components was also observed after 50°C treatment. This sensitivity was indicated by a reduction in the lytic activity for sensitized SRBC. Treatment for 15 minutes at 56°C with either RbRBC or SRBC was sufficient to abolish hemolytic activity in all equine sera tested. Chelation of cations with 0.04 M EDTA blocked expression of alternative and classical pathway activation; however, chelation of Ca⁺⁺ ions with 10 mM EGTA containing 1 mM Mg⁺⁺ ions permitted lysis of the RbRBC but not the SRBC. A dose-related Mg⁺⁺-ion dependence for RbRBC hemolytic activity was observed as the concentration of Mg⁺⁺ was increased to 1.0 mM. In addition, our results obtained with pre-colostral foal serum strongly suggest that natural antibody to RbRBC was of little importance in the lysis observed with these cells. These results also show that the equine alternative pathway activation may require Ca⁺⁺ ions. If Ca⁺⁺ ions are required, the equine alternative pathway is quite different from any other mammalian complement system so far described. Our results suggest that the alternative pathway of activation is of major importance in the equine complement system. Confirmation of this hypothesis requires both purification of the components involved as well as further characterization.     

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.     

Distinct serotypes of porcine rotavirus associated with diarrhea in suckling piglets in southern Quebec.

Cytopathic rotavirus strains were isolated in cell cultures from the intestinal contents of diarrheic piglets on Quebec pig farms where repeated outbreaks of enteritis occurred. All the isolates shared the common group antigens of rotaviruses as revealed by immunofluorescence and counterimmunoelectrophoresis. A hemagglutinating activity was demonstrated with human group O, porcine and guinea pig erythrocytes. At least one of the isolates was clearly distinguished from the American prototype of porcine rotavirus (strain OSU) by neutralization and hemagglutination inhibition tests; a third serotype was also suspected. By polyacrylamide gel electrophoresis of RNA, it was not possible to differentiate these isolates.     

A STANDARDISED HAEMAGGLUTINATION INHIBITION TEST FOR PORCINE PARVOVIRUS ANTIBODY

Basic variables of the haemagglutination inhibition (HI) test for porcine parvovirus antibody were investigated. Nonspecific serum inhibitors were satisfactorily removed without loss of specific antibody when undiluted serum was adsorbed with 25 percent kaolin in borate saline at pH 9.0. Natural haemagglutinins in test serums could be completely removed using 0.1 ml of packed erythrocytes to 0.6 ml of kaolin treated serums. Adsorption of prediluted serum resulted in a depression of specific antibody titres. Highest HI titres were obtained using guinea pig erythrocytes, following incubation of virus-serum mixtures for 18 hours at 4 degrees C, 3 hours at 25 degrees C or 2 hours at 37 degrees C. Micro- and macro-tests gave comparable HI titres.     

Haemagglutinating and hydrophobic properties of Corynebacterium (Eubacterium) suis

Corynebacterium (Eubacterium) suis strains from boars and sows haemagglutinated erythrocytes of different animal species (calf, guinea pig, poultry, pig, and human).The haemaigglutination was man nose resistant (MR) and was neither inhibited by L-fucose nor D-galactose. The hydrophobicity measured by salt aggregation test (0.1–0.9 mol/1 (NH₄)₂SO₄) and the hydrophobic interaction chromatography test (90 % retention in octyl sepharose) together with the haemagglutinating activity, indicated the presence of fimbriae on the bacteria. The haemagglutinating and hydrophobic properties were heat-sensitive (60°C for 10 min) suggestive of the presence of a protein structure. Two types of fimbria-tion were demonstrated by electron microscopy. Fetuin and glyco^ protein inhibited the haemagglutination, whereas porcine mucin was without any effect. These results indicate that branched glycoproteins might be important receptors for these fimbriae.The pathogenic aspects of C. suis are discussed, based on recent acquired knowledge of the effect of other pyelonephritogenic bacteria.     

Cell surface markers of the canine natural killer (NK) cell

Studies were performed to determine which of several cell surface markers are expressed on canine peripheral blood leukocyte (PBL) natural killer (NK) cells. Chromium-51 release assays showed a decrease in NK activity after depletion of PBL by carbonyl iron ingestion and adherence to IgG-antibody-coated ovine erythrocytes (EA gamma) and to IgM-antibody-complement-coated ovine erythrocytes (EA mu C). Effector cell adherence to and subsequent lysis of canine thyroid adenocarcinoma (CTAC) target cell monolayers provided direct visual identification of the putative canine NK cell. These surface immunoglobulin-negative cells, individually identified by their physical adherence to dead CTAC target cells, failed to form nonimmune rosettes with guinea pig erythrocytes or rosettes with EA mu or EA mu C. However, 39.0 +/- 4.2% of these adherent cells formed rosettes with EA gamma and 73.3 +/- 0.8% expressed the canine T-lymphocyte marker, Thy-1.     

Elucidating the role of ApxI in hemolysis and cellular damage by using a novel apxIA mutant of Actinobacillus pleuropneumoniae serotype 10

Exotoxins produced by Actinobacillus (A.) pleuropneumoniae (Apx) play major roles in the pathogenesis of pleuropneumonia in swine. This study investigated the role of ApxI in hemolysis and cellular damage using a novel apxIA mutant, ApxIA336, which was developed from the parental strain A. pleuropneumoniae serotype 10 that produces only ApxI in vitro. The genotype of ApxIA336 was confirmed by PCR, Southern blotting, and gene sequencing. Exotoxin preparation derived from ApxIA336 was analyzed for its bioactivity towards porcine erythrocytes and alveolar macrophages. Analysis results indicated that ApxIA336 contained a kanamycin-resistant cassette inserted immediately after 1005 bp of the apxIA gene. Phenotype analysis of ApxIA336 revealed no difference in the growth rate as compared to the parental strain. Meanwhile, ApxI production was abolished in the bacterial culture supernatant, i.e. exotoxin preparation. The inability of ApxIA336 to produce ApxI corresponded to the loss of hemolytic and cytotoxic bioactivity in exotoxin preparation, as demonstrated by hemolysis, lactate dehydrogenase release, mitochondrial activity, and apoptosis assays. Additionally, the virulence of ApxIA336 appeared to be attenuated by 15-fold in BALB/c mice. Collectively, ApxI, but not other components in the exotoxin preparation of A. pleuropneumoniae serotype 10, was responsible for the hemolytic and cytotoxic effects on porcine erythrocytes and alveolar macrophages.