Sheep Erythrocyte Preparation for Hemolytic Tests Exploring Complement Functional Activities.

Sheep erythrocytes (SE) are commonly used in complement functional tests. Non sensitized SE are useful to study the FH activity of cell protection. Indeed, as the cell surface of sheep erythrocytes is rich in sialic acids, Factor H (FH) is able to bind on it and therefore they represent a model of nonactivating surface. Because of their high capacity of complement regulation SE need to be modified to explore other functionality of the complement pathways, like the Complement hemolytic 50 (CH50) or the AP C3 convertase decay assays. For these tests, SE are sensitized with an anti-sheep red blood cell stroma antibody. In presence of serum or plasma complement components, sensitized SE may initiate complement cascade activation via the classic pathway explored in the CH50 assay. Sensitized SE may also be used to prepare C3b-coated SE that, with the use of buffers favoring AP, are suitable for the C3 Nef hemolytic assay and for the hemolytic assay studying the AP decay activity of FH. In this chapter we describe how to prepare SE for these different hemolytic tests.

Edwardsiella tarda evades serum killing by preventing complement activation via the alternative pathway.

Edwardsiella tarda is a Gram-negative bacterium with a broad host range that includes a wide variety of farmed fish as well as humans. E. tarda has long been known to be able to survive in host serum, but the relevant mechanism is unclear. In this study, we investigated the fundamental question, i.e. whether E. tarda activated serum complement or not. We found that (i) when incubated with flounder serum, E. tarda exhibited a high survival rate (87.6%), which was slightly but significantly reduced in the presence of Mg(2+); (ii) E. tarda-incubated serum possessed strong hemolytic activity and bactericidal activity, (iii) compared to the serum incubated with a complement-sensitive laboratory Escherichia coli strain, E. tarda-incubated serum exhibited much less chemotactic activity, (iv) in contrast to the serum incubated with live E. tarda, the serum incubated with heat-inactivated E. tarda exhibited no apparent hemolytic capacity. Taken together, these results indicate for the first time that E. tarda circumvents serum attack by preventing, to a large extent, complement activation via the alternative pathway, and that heat-labile surface structures likely play an essential role in the complement evasion of E. tarda.

Innate immune response of silver catfish (Rhamdia quelen) exposed to atrazine.

The impact of agrichemicals on aquatic vertebrate species has been a matter of increasing concern to researchers and environmentalist. In the present study, we evaluated the effects of a sublethal concentration of atrazine (10% of the LC(50-96 h)), a world-wide used herbicide, on the innate immune system of silver catfish (Rhamdia quelen). A significant reduction on phagocytic index, bacteria agglutination and bactericidal activity of the serum, serum lysozyme and total serum peroxidase activity was observed in fish exposed to atrazine for 24 h. After 10 days exposure to atrazine, only bactericidal activity of the serum, bacteria agglutination and total serum peroxidase activity were significantly reduced. Atrazine had no effect on the natural complement hemolytic activity. Our results demonstrate that atrazine decreases the innate immune response of fingerlings, which might increase its susceptibility to opportunistic pathogens.

The complement system of the goat: haemolytic assays and isolation of major proteins.

BACKGROUND: The aim of the present study was to develop a haemolytic assay for the study of the complement system in dairy goats (Capra aegagrus hircus) and to characterize the major goat complement system proteins. RESULTS: The commonly used sheep erythrocyte sensitized with rabbit antibodies were not sensitive to lysis by goat serum, but the combination of human red blood cells (RBC) plus rabbit antibodies was the best option found for goat complement assay. A buffer based on HEPES instead of the classical veronal (barbitone) was developed. Three proteins were isolated: factor H, C1q and C3 and these were compared with the corresponding human proteins. A novel affinity chromatography technique was developed for isolation of factor H. CONCLUSIONS: Human RBC plus rabbit antibodies were a suitable option for haemolytic assays. The isolated proteins are similar to the human counterparts.

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 ß-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.

Altered hematological and immunological parameters in silver catfish (Rhamdia quelen) following short term exposure to sublethal concentration of glyphosate.

Using agrichemicals to control unwanted species has become a necessary and common worldwide practice to improve crop production. Although most currently used agrichemicals are considered relatively safe, continuous usage contributes for soil and water contamination and collateral toxic effects on aquatic species. Few studies correlated the presence of agrichemicals on fish blood cells and natural immune system. Thus, in this study, silver catfish (Rhamdia quelen) were exposed to sublethal concentrations (10% of the LC(50-96 h)) of a glyphosate based herbicide and hematological and natural immune system parameters were evaluated. Silver catfish fingerlings exposed to glyphosate for 96 h had a significant reduction on blood erythrocytes, thrombocytes, lymphocytes and total leukocytes in contrast to a significant increase in the number of immature circulating cells. The effect of glyphosate on natural immune system was evaluated after 24h or 10 days exposure by measuring the phagocytic index of coelomic cells, and lysozyme, total peroxidase, bacteria agglutination, bactericidal activity and natural complement hemolytic activity in the serum of fingerlings. A significant reduction on phagocytic index, serum bacteria agglutination and total peroxidase was observed only after 24h exposure to glyphosate. In contrast, fingerlings exposed to glyphosate for 10 days had a significant lower serum bacteria agglutination and lysozyme activity. Glyphosate had no effect on serum bactericidal and complement natural hemolytic activity after 24h or 10 days exposure. Nonetheless, the information obtained in this study indicates that glyphosate contaminated water contributes to alter blood cells parameters and to reduce the activity of natural immune components important to mediate fish resistance to infecting microorganisms.

Pre-clinical evaluation of an sLe x-glycosylated complement inhibitory protein in a non-human primate model of reperfused stroke.

BACKGROUND: Soluble complement receptor-1 (sCR1), a potent complement inhibitor, confers neuroprotection in a murine stroke model. Additional neuroprotective benefit is achieved by sLe x-glycosylation of sCR1. In an effort to translate sCR1-sLe x to clinical trials, we evaluated this agent in a primate stroke model. METHODS: Adult male baboons randomly received either sCR1-sLe x or vehicle. Stroke volume was assessed on day 3, and neurological examinations were conducted daily. Complement activity (CH50) was measured at 30 minute, 2, 6, 12 hour, 3, and 10 days post-ischemia. RESULTS: The experiment was terminated prematurely following an interim analysis. In a preliminary cohort (n = 3 per arm), infarct volume was greater in the treated animals. No difference in neurological score was found between groups. CH50 levels were significantly reduced in the sCR1sLe x-treated groups. A hypotensive response was also observed in animals treated with sCR1-sLe x. Conclusions Further work is necessary to explain the hypotensive response observed in primates prior to further clinical development of sCR1-sLe x.

Haemolytic complement activity and humoral immune responses to sheep red blood cells in indigenous chickens and in eight German Dahlem Red chicken lines with different combinations of major genes (dwarf, naked neck and frizzled) of tropical interest.

A total of 376 chickens from different ecotypes were immunized with the non-pathogenic multi-determinant antigen sheep red blood cells (SRBC). The ecotypes included indigenous chickens from various locations in Tanzania (n=102), India (n=86) and Bolivia (n=89). In addition, eight German Dahlem Red (GDR) chicken lines with different major genes (dwarf, naked neck and frizzled) of tropical interest were also immunized with SRBC. Immune competence of the breeds was assessed by measuring complement haemolytic activity, both from the classical calcium-dependent complement pathway (CPW) and alternative calcium-independent complement pathway (APW), alongside IgTotal, IgG and IgM antibody responses to SRBC at 7 days post immunization. Large variations in complement activity and antibody responses to SRBC were observed within and between the indigenous breeds. Many indigenous chickens, especially from Bolivia, showed decreased complement activity (APW) following immunization with SRBC. Breeds from India showed the highest CPW activity and humoral (especially IgM) responses to SRBC, suggesting high immune competence. In contrast, Bolivian chickens were characterized by low CPW activity, low APW activity and low antibody levels to SRBC suggesting an overall low immune competence. In the GDR chickens, characterized by high CPW activity and high IgG antibody responses to SRBC, the major genes for naked neck, frizzling and dwarfism had no significant effect on the antibody responses and complement activity to SRBC.

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 degrees 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 Mg(2+) 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.

Association of the porcine C3 gene with haemolytic complement activity in the pig.

The complement component C3 plays an essential role in the activated complement system, which is involved in phagocytosis, inflammation and immunoregulation to destroy infectious microorganisms. The C3 molecule has more implications in the general defence mechanisms. In this study, the porcine C3 cDNA sequences including 5'- and 3'- flanking regions were determined and the polymorphisms in this gene were identified to carry out an association analysis between C3 and complement activity traits. Porcine C3 gene has high homology with human C3. Five single nucleotide polymorphisms (SNPs) and one microsatellite were detected in the porcine C3 gene. Haemolytic complement activity of alternative and classical pathways (ACH, CCP) was measured in 416 F2 animals of a crossbred of Duroc x Berlin Miniature Pig, which were immunized with Mycoplasma, Aujeszky and PRRS vaccines. C3 markers were found to be significantly associated (P <0.05) with both ACP and CCP. Animals with the more frequent haplotype present in Duroc and other commercial breeds exhibit higher ACP and CCP levels than the animals with haplotype specific to some Berlin Miniature Pigs. The association of C3 with complement activity reinforces the importance of C3 as a candidate gene for natural resistance to microorganisms.

Low vitamin E in diet reduces stress resistance of gilthead seabream (Sparus aurata) juveniles.

This study investigates the effect of dietary vitamin E on juveniles of gilthead seabream under stressful situations, focusing on the effects on growth, haematology, some immune parameters and plasma cortisol as indicators of stress. Two sardine meal-based experimental diets, one of them supplemented with 150 mg of alpha tocopherol kg(-1) of diet (control) and another one without vitamin E supplementation (diet NE), were assayed under two different stress conditions: overcrowding as a chronic stressor (during 15 weeks) and repetitive chasing as an acute repetitive stressor. Low levels of vitamin E in the diet depleted alternative complement pathway activity [from 167.23 U ml(-1) (control fish) down to 100.99 U ml(-1)] and also nonspecific haemagglutination. Also, fish fed a non-supplemented diet showed an elevation of plasma cortisol basal levels without a stressor influence [from 3.91 ng cortisol ml(-1) plasma (control fish) up to 21.70 ng cortisol ml(-1) plasma]. Low levels of vitamin E in the diet also produced an increase of erythrocyte fragility. Under chronic stress, fish fed the vitamin E-deficient diet showed a reduction in growth and survival, and alterations in haematological parameters, such as an additional haemoconcentration in response to overcrowding when compared with control fish. Under repetitive stress, fish fed the vitamin E deficient diet showed faster elevation of plasma cortisol levels in response to stress and a lower survival rate than control fish. Production of oxygen radicals by blood neutrophils was reduced under repetitive stress in fish fed the non-supplemented diet. These results suggest that fish fed the vitamin E-deficient diet had lower stress resistance.

Mechanism of serum resistance among Brucella abortus isolates.

It was shown in this study that complement-resistant Brucella abortus used were unable to activate complement in the absence of specific antibody. Complement-resistant isolates possessed O-antigen, but complement-sensitive organisms used are O-antigen deficient. Since B. abortus LPS does not activate the alternative pathway of complement, we concluded that activation of bovine complement must be due to some other mechanism. In this study, it was shown that bovine C1 binds to the outer membrane proteins of B. abortus. Isolated outer membrane proteins of both smooth (O-antigen positive) and rough (O-antigen negative) B. abortus used bind to C1q. However, only rough isolates were killed by complement. All of the O-antigen positive B. abortus isolates were complement-resistant. We propose that O-antigen shields outer membrane proteins and blocks C1q binding.

Studies on the haemolytic complement of the dromedary camel (Camelus dromedarius). I. Classical pathway haemolytic activity in serum.

Classical pathway haemolytic complement (CPHC) of the dromedary was assayed under standardised conditions. A total of 14 indicator systems of red blood cells (RBC) and haemolysins were investigated. Highest CH50 titre was obtained with rabbit RBC sensitised with goat haemolysin. Among the factors investigated were: ionic strength, Mg2+, Ca2+, ethylenediaminetetraacetic acid (EDTA) concentration, pH, incubation time and temperature. The standard system of titrating the HC levels consisted of rabbit RBC sensitised with goat haemolysin, sucrose veronal buffer (SVBS) pH 7.4, ionic strength 0.14 M and Ca2+ and Mg2+ concentrations of 4.0 x 10(-4) M and 1 x 10(-3) M, respectively. Incubation at 37 degrees C for 120 min gave the highest HC activity. Using these standardised conditions HC levels were determined in 79 camels aged between 3 months and 15 years. Highest mean HC value of 873 +/- 26.6 CH50 units ml-1 were recorded in the age group of 1-5 year old camels and the lowest mean HC value of 598 +/- 120.8 CH50 units ml-1 in the age group of 10-15 year old camels. Adult males in the age group 5-10 years had significantly higher mean HC levels than their female counterparts (P < 0.0001).

Natural hemolytic and bactericidal activities of sea bream Sparus aurata serum are effected by the alternative complement pathway.

Sea bream serum displayed bactericidal and hemolytic activities. These activities were depleted when serum was incubated with different activators of the alternative complement pathway (ACP). Ethylenediaminetetraacetic acid (EDTA) inhibited both the hemolytic and bactericidal activities, while ethyleneglycol-bis (B-aminoethyl ether)-N, N, N'-tetraacetic acid (EGTA) was not inhibitory. An antibody against the putative third component of sea bream component (C3) was produced. It was observed by immunoelectrophoresis that the sea bream C3 and human C3 migrated in the same position. Crossed immunoelectrophoresis showed that sea bream C3 exhibited a similar pattern of activation when compared with its human counterpart. The anti-sea bream C3 antibody inhibited both bactericidal and hemolytic activities. It was concluded that both serum actions were displayed by the ACP. The best conditions for the sea bream ACP titration were investigated. Of all mammal erythrocytes tested, rabbit erythrocytes (RaRBC) were found to be the best ACP activators and thus were used for the titration. Sea bream showed very high ACP titers when compared with those of mammals. Absorption of naturally occurring antibodies against rabbit RaRBC did not influence the ACP titers. Enzymatic removal of sialic acid from different mammalian erythrocytes increased the sensitivity of these cells to hemolysis mediated by the sea bream ACP.

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.

Determination of optimal in vitro conditions for caprine alternative complement pathway assay.

Optimal in vitro testing conditions for caprine alternative complement pathway assay were determined. Effects of the following variables were tested: heterologous erythrocytes; pH, ionic strength and Mg2+ ion concentration of the complement diluent; incubation time and temperature. Rabbit erythrocytes were the optimal target cells. The optimal buffer conditions were: pH 8.0, ionic strength 0.06 mmol NaCl and 5mmol Mg2+ ion. Optimal incubation time and temperature were 75 min and 30 degrees C, respectively.

A new hemolytic assay for bovine serum complement and its application during experimental bovine anaplasmosis.

A new hemolytic assay for bovine complement is presented. Using this assay we found a significant reduction in bovine serum complement activity during the acute phase of anaplasmosis, and an increase in the sensitivity of the red blood cells (RBC) to bovine complement lysis in vitro. The new hemolytic test is performed with bovine RBC, rabbit anti-bovine RBC serum and bovine serum complement. An isotonic sucrose Tris-buffered saline solution of ionic strength 0.094 and pH 7.2 was found to be adequate for this test. The titres obtained with this new assay, which uses autologous RBC, are comparable with those obtained using the guinea pig RBC assay. The finding of a reduction in bovine serum complement during anaplasmosis may be suggestive of a mechanism responsible for the pathology of this disease.