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.     

Bovine IgG2a antibodies to Haemophilus somnus and allotype expression.

Bovine IgG2a has been implicated in protection against pyogenic infections, including those caused by Haemophilus somnus. To further investigate the role of IgG2a in defense against H. somnus, IgG1 and IgG2a antibodies were purified from antiserum against an immunodominant 40 kDa outer membrane protein (p40) of H. somnus, which was previously shown to passively protect calves against H. somnus pneumonia. The passive protective capacity of anti-p40 IgG1 or IgG2a was evaluated in vivo in calves. Purified anti-p40 IgG1 or IgG2a was incubated with H. somnus for 15 min before intrabronchial inoculation of calves. Bacteria incubated with anti-p40 IgG1 or IgG2a were inoculated into one caudal lung lobe and bacteria incubated with IgG1 or IgG2a from the respective preimmunization serum were inoculated into the contralateral lobe. The volumes of pneumonia in the right and left lungs were determined 24 h later. The difference in volume of pneumonia with H. somnus preincubated in IgG1 pre- and postimmunization anti p40 was less (16 cm3, P = 0.298) than the difference in volume of pneumonia with H. somnus preincubated in IgG2a pre- and postimmunization anti p40 (30 cm3, P = 0.146). Although the differences in lesion size between pre- and postimmunization serum were not statistically significant, the trend suggests IgG2a may be more protective than IgG1. To examine this further, the peptide specificity of these IgG1 and IgG2a antibodies to p40 was examined. After limited proteolysis of p40, IgG2a antibodies reacted with 2 peptides not recognized by IgG1 antibodies. Other peptides were recognized by both isotypes. Since these studies suggested that IgG2a may be important in protection against infection, we then investigated some aspects of the role of the 2 IgG2a allotypes, A1 and A2. In retrospective studies of age differences in expression of IgG2a allotypes, no heterozygotes were detected in calves of 60 d old or less, and fewer heterozygotes were detected in calves 61-120 d old than in cattle older than 270 d (P < 0.01). In a subsequent prospective study of the time course of allotype expression, Holstein calves shown to be heterozygotes expressed the IgG2aA1 allotype early but the IgG2aA2 allotype was not usually detected until 3 to 4 mo of age. Thus, both the retrospective and the prospective studies showed age related differences in expression of the IgG2aA1 and A2 allotypes. This could have implication in protection.     

Activation of complement by bovine respiratory syncytial virus-infected cells

Because complement activation is probably involved in the pathogenesis of as well as in recovery from the disease induced by bovine respiratory syncytial virus (BRSV), we studied the activation of complement by BRSV-infected cells in vitro in a homologous system. Binding of C3 on the surface of infected cells was measured in a biotin-streptavidin amplified ELISA, and complement-mediated lysis was measured in a 51Cr release assay. Without antibody, infected cells activated and bound more C3 than uninfected cells. C3 activation that occurred in the absence of antibody was largely mediated by the classical pathway and induced lysis inefficiently. BRSV-specific antibody enhanced complement activation as measured by both C3 ELISA and cytotoxicity assay. In the presence of antibody, C3 activation was largely dependent on the alternative pathway and efficiently induced lysis. Both IgG1 and IgM antibodies enhanced C3 activation, but IgG2 and IgA did not enhance C3 activation in our experiments. Preincubating cells with IgA or IgG2 did not inhibit C3 activation enhanced by IgG1 or IgM. Murine monoclonal IgG1 antibodies against epitopes on the Fusion protein of the virus also enhanced C3 binding, but differed in their capacity to induce complement-mediated lysis.     

Cloning and characterization of cDNAs encoding four different canine immunoglobulin γ chains

cDNAs encoding four different canine immunoglobulin G (caIgG) γ chains were identified in this study. One of these IgG γ chain cDNAs, (caIgG-A), represents 92.5% of the IgG γ chain cDNAs in a dog spleen cell cDNA library; a second partial IgG γ chain cDNA (caIgG-B) was also identified in the library. The other two IgG γ chain cDNAs (caIgG-C and caIgG-D) were RT-PCR amplified from canine lymphoma samples. Comparison of the four different canine IgG γ chain cDNAs showed homologies from 83.6 to 89.2% and from 73.1 to 81.8% at nucleotide and amino acid sequence levels, respectively. Despite the high similarity in CH1, CH2 and CH3 domains among the different caIgG γ chains, the hinge regions were distinct, sharing only 19.0–35.2% homology at the amino acid level. No multiple duplication of the hinge region, as reported for human IgG1 and IgG3, was detected in any of the canine IgG γ chains. The numbers of cysteines in the putative hinge regions were found to be 3, 2, 7 and 3 for the four canine IgG heavy γ chains (A, B, C and D), respectively. Specific primers were designed based on caIgG γ chain hinge region DNA sequences and were used in RT-PCR for measuring different caIgG γ chain mRNA levels in canine PBMC samples.     

Mycoplasmacidal action of bovine complement mediated by bovine IgG1, IgG2 or IgM

The effect of several inhibitors of complement were examined in haemolytic, bactericidal and myoplasmacidal systems with bovine serum as the complement source. It appears that although EGTA-Mg allows the alternative complement pathway to function in a haemolytic system it has an inhibitory effect on this pathway in bactericidal and mycoplasmacidal systems. Both ?-aminocaproic acid and salcyladoxime were found to be useful for distinguishing the complement pathways in bovine serum and the results of experiments with these substances indicated that bovine IgG1 and IgG2 activated bovine complement, with a mycoplasma as the target cell, by the classical pathway. Mycoplasma bovis, which unlike Acholeplasma laidlawii, does not activate the alternate pathway in gnotobiotic-calf serum, was killed by serum from cattle that had not been infected previously with this mycoplasma. In this case killing was apparently mediated by cross-reacting IgM and complement via the classical pathway.     

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.     

Polymorphism of the CD4 and CD5 differentiation antigens in cattle.

Monoclonal antibodies (mAbs) specific for bovine CD4 and CD5 antigens have been found to identify polymorphic determinants on these molecules. In the case of CD5, mAb IL-A67 recognises one allotypic form of the antigen while four other CD5-specific mAbs in the workshop (CC17, CC29, BLT-1 and 8C11) recognise a second allotype. The CD4-specific mAbs submitted to the workshop reacted with the cells of all animals tested. However, a further two mAbs (CC26 and IL-A18) specific for CD4 were found to react with cells only from about 85% of animals tested. Sequential immuno-precipitation experiments together with family studies showed that the allotypes of CD4 and CD5 are both inherited in a simple Mendelian manner and are co-dominantly expressed. One of the CD5 allotypes was not detected in Bos taurus animals while the gene frequency of the second allotype was only about 10% in the B. indicus animals tested. The gene frequency of the CD4 allotype detected by CC26 and IL-A18 was similar in the two sub-species.     

Isolation of bovine complement factor H

A bovine serum protein, initially recognized by its inhibitory effect on the hemolytic activity of the bovine alternative pathway was isolated from fresh bovine serum by polyethylene glycol precipitation and chromatography on DEAE-Sephacel, CM-Sephadex A-50 and Sephadex G-200. The protein, a single chain polypeptide with an apparent molecular weight of 158,000, was identified as factor H, a regulatory protein of the alternative complement pathway. Functional characterization of this protein as factor H was based on the following properties: binding to C3b, inhibition of factor B binding to C3b, cofactor activity in the cleavage of C3b by factor I, inhibition of fluid phase alternative pathway C3 convertase (C3b.Bb) formation and activity, and species-specific inhibition of the alternative pathway mediated hemolysis of heterologous erythrocytes. A monospecific rabbit antiserum against bovine factor H failed to react with human serum factor H.     

A sensitive microassay for the determination of hemolytic complement activity in bovine milk

A ⁵¹Cr release microhemolytic complement assay is described to detect hemolytic complement activity in bovine milk. ⁵¹Cr-labeled guinea-pig erythrocytes (GPRBC), which have been sensitized with a subagglutinating amount of rabbit anti-GPRBC, are placed in microtiter plates. Pooled bovine sera as source of complement to achieve about 50% of ⁵¹Cr release were added to each well prior to the addition of the samples on the test. Determination of CH100 titer was obtained by difference of counting between heated and unheated diluted whey samples from a standard linear regression. Comparative hemolytic values throughout lactation were established for the first time and confirmed the improved sensitivity of the assay.     

Alternate complement pathway in porcine sera: Lysis of guinea pig erythrocytes

The hemolysis by porcine sera of unsensitized erythrocytes (EU) from nine different species was investigated. Optimal lysis occurred when porcine sera were reacted with unsensitized guinea pig erythrocytes suspended in a pH 6.5, barbital-buffered saline solution, made 0.1% in gelatin, and containing 10 mm ethyleneglycol-bis (β-amino-ethyl ether) N, N¹-tetraacetic acid and 4 mm MgCl₂ (BSG-EGTA-Mg). Results of studies with several different treatments that inhibit complement (C) induced hemolysis indicated that the alternate C pathway was involved in the lysis of EU in the BSG-EGTA-Mg buffer. The extent of lysis was decreased when porcine sera were adsorbed with zymosan, mixed with 20 mm salicylaldoxime, or heated at 50%C. However, carrangeenan treatment caused only a slight decrease in the extent of hemolysis induced. Cobra venom factor activated the alternate C pathway in porcine sera. The pattern of C component utilization resulting from lysis of EU by porcine sera indicated activation of the alternate and not the classical C pathway. Extensive adsorption of porcine sera with packed guinea pig erthrocytes at 0°C only slightly reduced its capacity to lyse guinea pig erythrocytes. Collectively, these results provided evidence that the membrane of the guinea pig erythrocyte is able to active the alternate C pathway of porcine sera without the direct involvement of specific antibody.     

Three novel single-nucleotide polymorphisms of complement component 4 gene (C4A) in Chinese Holstein cattle and their associations with milk performance traits and CH50

Complement component 4 (C4A) is a candidate gene that reflects complement activity. The primary role of this gene in the classical and lectin-activation pathways is to provide protection against bacterial pathogens. In the current study, the bovine complement C4A gene was screened for polymorphisms, and the associations of these polymorphisms with the hemolytic activity of the classical pathway (CH50), C4 serum levels, and milk performance traits were examined. Three novel single-nucleotide polymorphisms (rs 132741478: g.2994 A>G, rs 134006517: g.3508 A>G, and rs 137485678: g.3649 G>C) were detected by DNA sequencing and PCR-RFLP in 1182 Chinese Holstein cows. The rs 132741478: g.2994 A>G mutation in exon 10 led to methionine and valine exchange at position 362, whereas rs 134006517: g.3508 A>G and rs 137485678: g.3649 G>C were synonymous substitutions. The statistical analyses revealed that cows with rs 132741478: g.2994 A>G-AG and rs 137485678: g.3649 G>C-CC have significantly lower somatic cell scores (SCS, P<0.01). Homozygote cows with GAC haplotypes have the lowest SCS, whereas AAG/AAC cows have the highest. The serum concentration of C4 by ELISA and the hemolytic and antibacterial activity of CH50 were also evaluated in the current study. The results confirmed that rs 132741478: g.2994 A>G in the coding sequence of the β-chain of the bovine C4A gene is related to mastitis resistance. This polymorphism may be very important in marker-assisted selections in dairy cattle breeding programs.     

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.     

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.     

A simple hemolytic assay for bovine complement component C3

A simple, one-step, alternative pathway (AP) hemolytic assay for bovine C3 has been developed. Methylamine was used to prepare a bovine serum reagent, R3, functionally depleted of C3. The addition of purified bovine C3 to the R3 reconstituted, in a dose-dependent manner, the hemolytic activity for unsensitized heterologous erythrocytes. The assay was used to determine relative levels of C3 in different bovine serum samples. Human C3 and bovine C3 were interchangeable in the assay. Reconstitution of bovine and human R3 reagents with homologous or heterologous C3, in the presence of different species of erythrocytes, provided evidence that cell surface regulation of the homologous hemolytic AP may not be limited to the assembly and activity of the C3 convertase. The AP assay was more sensitive and less complex to perform than a standard classical pathway assay for bovine C3.     

Opsonization of yeast cells with equine iC3b, C3b, and IgG

The main opsonins in serum are antibodies and complement factor C3. The opsonization mechanisms including complement activation and deposition are important in studies of phagocytosis and of mechanisms of microbial immune evasion. The objective of the present study was to monitor the deposition of complement C3 and IgG from equine serum on yeast cells (Saccharomyces cerevisiae) using a flow cytometric immunoassay. Correlations were made between the opsonic coating and phagocytic capacity using equine blood neutrophils. In addition, the bound C3 fragments were characterized by SDS–PAGE and Western blot analyses.

Opsonic coating of yeast with equine C3 and IgG occurred rapidly with detectable levels with as little as 0.75% serum. C3 deposition was a result of complement activation and no passive adsorption was observed. When complement was inactivated, the fluorescence indicating IgG deposition increased 3–6-fold, indicating spatial competition between C3 and IgG at binding.

Opsonization with 1.5% serum led to suboptimal equine neutrophil phagocytosis of yeast cells which was dependent on complement activation by the classical pathway. With ≥6.25% serum, IgG contributed to opsonization and phagocytosis. With 50% serum and more, C3 was deposited also by the alternative pathway. Phagocytosis rates became optimal with 3% serum, and did not increase further with higher serum concentrations. The main form of C3 on the yeast cells was iC3b and the rest was C3b without any detectable breakdown products (C3c or C3dg). The equine complement components are similar in size to the human equivalents.

It may be concluded that opsonization of yeast particles leading to phagocytosis, occurs at very low serum concentrations (1.5%) and that it is dependent on activation of the classical complement pathway at this low opsonic level. This is an important finding for efficient host defense, e.g. extravascular phagocytosis at infection sites.     

Development of ELISAs for the measurement of IgM and IgG subclasses in sera from llamas (Lama glama) and assessment of the humoral immune response against different antigens

Members of the Camelidae family possess a functional class of antibodies devoid of light chains (known as heavy chain antibodies, HCAbs). Three IgG isotypes have been identified (IgG(1), IgG(2) and IgG(3)); IgG(2) and IgG(3) are HCAbs whereas the IgG(1) has the conventional structure. Different subtypes of IgG(1) (IgG(1a) and IgG(1b)) and IgG(2) (IgG(2a), IgG(2b) and IgG(2c)) have been classified according to variations in the amino acids sequence of the hinge region. The single variable domain of HCAbs has been referred as VHH. Until now, the relative amount of each subclass has been inferred, but the lack of highly specific antibodies against HCAbs has been a limitation for their quantification. In a previous work, we produced specific polyclonal antibodies against IgG(2a), IgG(2b), IgG(2c) and IgG(3) by immunizing rabbits with synthetic and recombinant peptides corresponding to their hinge region. In this work we produced specific antisera against llama IgM and IgG(1). The anti-IgG(1) serum was obtained by immunizing rabbits with a recombinant fusion protein formed by GST fused to the CH(1) domain of the IgG(1). The anti-IgM serum was obtained by immunizing rabbits with IgM heavy chain. All these antisera were useful for the development of ELISAs for the measurement of IgM, total IgG and IgG subclasses. Sera from llamas (n=20) analyzed by ELISA gave the following values of immunoglobulins: IgG(1)=6.168+/-1.628 mg/ml; IgG(2)=0.684+/-0.310 mg/ml; IgG(3)=1.232+/-0.410 mg/ml; total IgG=8.933+/-1.815 mg/ml and IgM=1.027+/-0.308 mg/ml. These results indicate that HCAbs represent almost 25% of total IgG and the IgG(3) subtype is the predominant HCAb. We also analyzed the primary humoral immune response after immunization llamas with different antigens (BSA, BSA-DNP and dextran). Although it has been described that a few VHH clones are very efficient in the interaction with haptens, in this case the response against DNP was characterized by a delayed appearance of HCAbs in comparison with that of IgG(1). No anti-dextran response was observed in any of the isotypes analyzed.     

Isolation and characterization of the eighth component of the bovine complement system

OBJECTIVE: To isolate and characterize the eighth component of the complement system (C8) in cattle. SAMPLE POPULATION: Fresh plasma obtained from beef cattle. PROCEDURES: Plasma samples were fractionated, using sequential precipitation and ion-exchange and gel-filtration chromatography, to yield C8. The protein was identified throughout the procedure on the basis of its hemolytic function. Electrophoresis in polyacrylamide gels was used to determine molecular weight and composition of polypeptide chains. Reconstitution of classical and alternative complement pathways was used to characterize the hemolytic function of bovine C8. RESULTS: The bovine C8 protein consisted of a disulfide-bonded alpha-gamma heterodimer that was noncovalently associated with a beta chain. Apparent molecular weight of the alpha, beta, and gamma chains under reducing conditions were 66, 61, and 23 kd, respectively. In the classical pathway of activation, bovine C8 and the ninth component of the complement system (C9) had species incompatibility with human C8 and C9 on sheep erythrocyte target cells. CONCLUSIONS: A simple 4-step fractionation procedure provided good yield of bovine C8 from plasma. The isolated protein was structurally comparable to C8 from other species. Purified bovine C8 may be useful in functional hemolytic assays to investigate the roles of complement-mediated lysis in the pathogenesis of inflammatory diseases and the killing of susceptible microorganisms.     

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.     

The relationship between the variants of the bovine MBL2 gene and milk production traits, mastitis, serum MBL-C levels and complement activity

Mannose-binding lectin (MBL), a calcium-dependent collagenous lectin, plays an important role in the host immune defence against a wide range of pathogens. There are MBL1 and MBL2 genes which encode the MBL-A and MBL-C proteins, respectively. This study was carried out to investigate the relationship between the variants of the bovine MBL2 gene and milk production traits, mastitis, serum MBL-C levels and hemolytic complement activity in both classical pathway (CH50) and alternative pathway (ACH50) in Chinese Holstein cattle. Four single-nucleotide polymorphisms (SNPs) in the exon 1 of the MBL2 gene in Chinese Holstein cattle and Luxi yellow cattle were identified by the direct sequencing method. The SNP g.201 G>A was identified as a non-synonymous mutation (codon 31, Arg>Gln) at the N-terminus cysteine-rich domain and the SNPs g.234 C>A and g.235 G>A (codon 42) made Pro to Gln at the 1st Gly-X-Y repeat of the collagen-like domain, while the SNP g.244 T>C (codon 45) was identified as a synonymous mutation (Asn>Asn) at the 2th Gly-X-Y repeat of the collagen-like domain. The SNP markers (g.201 G>A, and g.234 C>A) were significantly correlated with somatic cell score (SCS) (P<0.05). The concentration of MBL-C protein in serum ranges from 0.8 to 7.4μg/mL by enzyme-linked immunosorbent assay. Six combinations of different haplotypes from the four SNPs were identified in Chinese Holstein cattle. Statistical analysis revealed that cows with the haplotype combination H4H5 exhibited the lowest SCS. The CH50 value of H4H5 and H5H5 cow are significantly higher than H2H5 haplotype combination (P<0.05). The association analysis results showed that the haplotype combination H4H5 may be used as a tolerance haplotype combination for the bovine mastitis.     

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.