Complement fixation is an immunological reaction involving the binding of complement proteins to antibodies that have attached to specific antigens. In horses, this process is part of the innate immune response, contributing to the identification and elimination of pathogens. The complement system consists of a series of proteins that, when activated, enhance the ability of antibodies and phagocytic cells to clear microbes and damaged cells. This system also promotes inflammation and attacks the pathogen's cell membrane. The complement fixation test is a diagnostic tool used to detect the presence of specific antigens or antibodies in equine serum by observing the consumption of complement proteins. This page compiles peer-reviewed research studies and scholarly articles that explore the mechanisms, regulation, and diagnostic applications of complement fixation in equine immunology.
Siegel RC, Cathou RE.Complexes of IgM equine anti-dansyl antibodies and different dansyl substituted carriers were tested for their ability to fix complement (C). Only dansyl92-Ficoll and dansyl12-poly-L-lysine were found to be effective. Dansyl13-bovine serum albumin, dansyl127-keyhole limpet hemocyanin, and reduced and alkylated dansyl10-ribonuclease were all ineffective. Lack of C fixation by the dansyl-ribonuclease was not due to lack of antibody-antigen complex formation, since binding at the concentrations employed for C fixation was established. However, in contrast, polymerized dansyl-ribonuclease (polydis...
Conner ME, Darlington RW.Fecal samples from 86 foals with diarrhea were examined by electron microscopy during a 2.5 year period. Of these, 26 (30%) were positive for rotavirus. All of the cases were found in epizootic areas. The disease was produced in an experimental foal by inoculation via stomach tube of a bacteria-free fecal filtrate containing rotavirus. Examination of postmortem tissues from the duodenum and jejunum of 2 naturally infected foals and an experimentally infected foal revealed replicating virus in the intestinal epithelial cells. A limited survey of complement-fixing antibody to rotavirus in horses...
Otani S, Arimitsu Y, Akama K.Antileptospiral sera from hyperimmunized horses were fractionated by gel filtration on Sephadex G-200 or by starch block electrophoresis. The fractions were examined quantitatively for leptospiricidal, agglutinating and complement fixing activities. The leptospiricidal activity was higher in the 78 globulin fraction than in the 19S globulin fraction, while the agglutinating activity was shared by both the fractions being higher in the 19S fraction. Complement fixing activity was found evenly in both the fractions. Leptospiricidal and complement fixing activities were higher in gamma-globulin t...
Donnelly J, Joyner LP, Graham-Jones O, Ellis CP.The incidence of antibodies to Babesia equi and B. caballi in horses in the Royal Stables of His Majesty the Sultan of Oman was assessed by complement fixation (CF) and immunofluorescent antibody (IFA) tests. Two series of samples taken with a 2-year interval, mainly from animals reared in Oman, indicated a stable but high prevalence of antibodies. On the 2 occasions 94.6 and 97.7% respectively were positive to B. equi by IFA and 76.8 and 75.0% were positive by CF. For B. caballi the corresponding percentage figures were lower--67.9 and 40.9 by IFA and 30.4 and 40.9 by CF. A group of animals t...
Sentsui H, Kono Y.A seroepidemiological survey was performed on antibody against Getah virus in horses in Japan by the complement fixation test. The positive rate was 35 and 53% in two areas where an outbreak of the infectious disease was reported, whereas it was in a range of 3.3 to 24.2% in other areas, except in certain prefectures of the Kyushu district where a high positive rate was observed. In the Hokkaido district, the northernmost part of Japan, no reactors were found in horses under 6 years old, unlike in any other district. It was also suggested that Getah virus infection might have already been prev...
Cinco Del Fabbro M, Dougan R, Jelincic A, Piacentini I.The macroagglutination test, according Mailloux, was investigated for its feasibility in the rapid diagnosis of human and animal leptospirosis. Suspected sera examinated by Mailloux test, were also examinated by Complement Fixation and Microagglutination; the results suggest that: Mailloux macroagglutination is the serological test of choice, for screening of animal and human sera, mostly if it is not needed to know the infecting serovar.
Timoney PJ, O'Reilly PJ, McArdle JF, Ward J, Harrington AM, McCormack R.Reproduction of contagious equine metritis 1977 in Pony mares was achieved with cultures of an unclassified Gram-negative coccobacillus. Infected mares developed a vaginal discharge and associated inflammatory changes of the cervix and vagina. There was evidence of variation in pathogenicity between different strains of the organism. Although all infected mares made spontaneous clinical recoveries, the Gram-negative coccobacillus persisted in the genital tracts of a considerable proportion for a variable period after challenge. Recovery of the organism was not associated solely with the occurr...
Tatarov G, Martinov S, Panova M.It was established that the complement binding reaction (CBR) is a suitable and very fast method for horse rhino-pneumonitis diagnostics. Cell cultural virus produced in cell cultures of pig kidneys was used as antigen. The antigen lots tested have no anticomplementary properties. Highest complement binding activity was evident in the non-diluted antigen, which discovered specific antibodies in immune serums. The CBR specificity was tested by the aid of homologous and heterologous serums and antigens. The titers of complement binding antibodies in the serums of 255 horses recovered from the di...
Stalheim OH, Stone SS, Blackburn BO, Foley J.In horses given whole cultures or cells of Mycoplasma mycoides subsp capri (by subcutaneous and intravenous injections), antibody responses were measured by serologic procedures. During an immunization period of 22 weeks, horses produced an antiserum that was used to identify M mycoides subsp capri by agglutination, complement-fixation, and fluorescent antibody (FA) tests, but not by the growth-inhibition test. Horses that were injected with whole cultures of M mycoides subsp capri responded better than horses that were injected with only cells, ie, antibodies were detectable sooner by agar ge...
Croxton-Smith P, Benson JA, Dawson FL, Powell DG.A complement fixation test (CFT) based on that used for brucellosis (Brinley Morgan and others 1971) has been developed for use on the sera of horses exposed to the contagious equine metritis (CEM) organism. None of 50 single samples from horses thought to be unexposed to the CEM organism was positive to the test, although five showed inconclusive reactions. Samples were examined from 41 mares either proved to be infected or from an infected stud. Of these 21 were positive, 11 were inconclusive and nine were negative. The relationship of the CFT to reactions in the other tests used in this con...
Dawson FL, Benson JA, Croxton-Smith P.Serum agglutination tests, anti-globulin tests, and complement fixation tests were carried out on sera taken over a period of 98 days from two fillies experimentally infected with the contagious equine metritis organism. The pattern, and significance in diagnosis, of these results is discussed. All 3 tests showed positive titres in the acute phase of experimental disease; reactions in the complement fixation test persisted longest.
Ferguson JA, Reeves WC, Milby MM, Hardy JL.Of 359 horses vaccinated with attenuated Venezuelan equine encephalomyelitis (VEE) vaccine (strain TC-83), 87% developed hemagglutination-inhibition (HI) antibodies to VEE virus within 1 month. Blood from a subsample of 101 of the 359 horses was obtained over a 1-year period. Within 1 month after vaccination, 84% of the 101 horses had developed VEE HI antibodies, 87% had developed VEE-neutralizing (Nt) antibodies, and 78% had developed VEE complement-fixing (CF) antibodies. One year after vaccination, 58% of the horses had VEE HI antibodies and 73% had VEE Nt antibodies. The percentage of hors...
Gutekunst DE, Malmquist WA, Becvar CS.Antiserums prepared in specific pathogen free (SPF) ponies were used in direct and indirect immunofluorescence, immunodiffusion, complement fixation and serum neutralization procedures to study the interrelationships of the three types of equine herpes viruses (EHV-1, EHV-2, and EHV-3). Equine cell cultures infected with each type virus fluoresced when stained with homologous conjugated antiserum. In reciprocal tests EHV-1 and EHV-3 cross-fluoresced, but EHV-2 did not cross-fluoresce. Non-infected cell cultures did not fluoresce when stained with the 3 conjugates. EHV-1 and EHV-3 cross-fluores...
Zakin MM, Peña C, Poskus E, Stewart JM, Paladini AC.The immunological properties of a synthetic human growth hormone fragment comprising the amino acids 73 through 128 and of the homologous natural horse growth fragment formed by amino acids 73 through 123, have been comparatively studied. Antisera obtained in rabbits inoculated with the native human hormone or with the fragments, were used. By hemagglutination experiments both fragments have the same reactivity toward the anti-human growth hormone serum, but complement fixation curves detect the existence of at least two populations of antibodies presumably originated against the sequence 73-1...
McGuire TC.Isolated equine immunoglobulin (Ig)G(T) antibodies to equine infectious anemia virus P26 antigen did not precipitate with antigen when the ratio of antibody to antigen was high. However, at lower ratios of antibody to antigen precipitation occurred. In addition, complement-fixation by IgG and P26 antigen was inhibited by high concentrations of IgG(T). The unusual reaction pattern noted with IgG(T) antibodies was still detectable by the immunodiffusion test for equine infectious anemia virus. In situations of nonprecipitability by IgG(T), the adjacent positive control line was inhibited, and th...
Benmansour A, Benelmouffok A, Bouguermouh A.During an epizootic of equine acute respiratory disease in Algeria, a strain of equine influenza virus was isolated. Sera examination by hemagglutinin inhibition test and complement fixation test confirmed the etiology of the disease. The first and second outbreak of the disease remained localised. The third outbreak spread within few months to all parts of the country. Horses vaccinated with a commercial equine influenza vaccine remained healthy.
Thouless ME, Bryden AS, Flewett TH, Woode GN, Bridger JC, Snodgrass DR, Herring JA.Human, piglet, mouse, foal, lamb, calf and rabbit rotaviruses all infected, but could not readily be subcultured in LLC MK2 cells. Cells infected with mouse and calf rotaviruses reacted by indirect immunofluorescence (FA) with convalescent serum from children, piglets, mice, foals, lambs, calves or rabbits, taken after rotavirus infection. Human, calf, piglet, mouse and foal rotaviruses reacted with human, calf, mouse, foal and lamb convalescent serum by complement fixation (CF). It was not possible to distinguish between different rotaviruses by CF or FA. Neutralization tests, however, detect...
Dinter Z, Klingeborn B.Six cases of paresis occurred in a Swedish stud with 48 mares and a stallion. Complement-fixation tests revealed a recent infection with EHV-1 in most horses of the stud. Serumneutralisation tests showed rapid antibody-titre increases during the course of the disease. This type of antibody response was interpreted as induced by reinfection or, possibly, recurrent infection. Two diseased mares were sacrificed. No virus could be isolated from their central nervous system (CNS), liver or spleen, but there is a presumptive evidence for the presence of an antigen specific to EHV-1 in the CNS and li...
Barban PS, Minaeva VM, Pantiukhina AN, Startseva MG.A comparative study was made of the serological properties and virus-neutralizing activity of antiencephalitis gamma-globulin and Fab-fragments isolated from it by gel-filtration. Horse immunoglobulins against the autumno-summer tick-borne encephalitis virus could be disintegrated with the aid of papaine to monovalent Fab-fragments which (according to the complement fixation reaction, the test of suppression of the complement fixation, and the HAIT) retained the serological activity whose level was compared with that of the serological activity of gamma-globulin. Fab-fragments possessed a mark...
Poskus E, Zakin MM, Fernámdez HN, Paladini AC.Peptide fragments, obtained from equine growth hormone by cyanogen bromide cleavage and further chemical treatment, were isolated and identified. Their immunological reactivities were tested by hemagglutination and complement fixation methods using rabbit antisera against native hormone. Antigenic determinants were detected in the fragments comprising amino acid sequences 5-72 and 73-123, this last one being predominant. Fragment 124-178 had very low reactivity. Nitration of peptide 73-123 did not modify its immunological properties,but oxidation diminished them. Comparison of the antigenicity...
Thomson GR, Mumford JA, Campbell J, Griffiths L, Clapham P.An investigation was made of 3 serological tests (virus neutralization, complement fixation and indirect immunofluorescence), which are applicable to epidemiological studies of infections by Equid herpesvirus 1 (EHV-1). Sera from gnotobiotic foals inoculated intranasally with various strains of EHV-1 were unable in some cases to neutralize heterologous strains and these results were not consistent with the existence of clearly-defined subtypes of EHV-1, as previously proposed. The cross-reactions in complement-fixation tests paralleled those with neutralization but immunofluorescence tests wer...
Blakeslee JR, Olsen RG, McAllister ES, Fassbender J, Dennis R.Five horses were experimentally exposed to equine herpesvirus 2 strain LK. Two young foals developed chronic pharyngitis (98 and 232 days, respectively). Growth characteristics, cytopathic effects (CPE), inclusion body formation, ether sensitivity, and immunofluorescent analysis indicated that the virus recovered from infected animals was a herpesvirus serologically identical with, or at least antigenically related to EHV-2 strain LK. No significant complement-fixing (CF) or virus-neutralizing (VN) antibody responses were observed in adult horses while both foals demonstrated a rise in CF anti...
Pascoe RR, Bagust TJ.Equine coital exanthema can be produced experimentally in stallions by inoculation with an equine herpesvirus (strain 65/61) and be transmitted during coitus with an infected mare. Serological responses to this infection include the production of complement-fixing and serum-neutralizing antibodies which reach maximum levels 14 to 21 days after infection. Complement-fixing antibodies decline rapidly and are usually not detectable by 60 days after infection, whereas serum-neutralizing antibody activity is maintained for at least 1 year. This disparity provides a useful method for the diagnosis o...
Barban PS, Minaeva VM, Pantiukhina AN, Startseva MG.A comparative study was made of the serological properties and virus-neutralizing activity of antiencephalitis gamma-globulin and Fab-fragments isolated from it by gel-filtration. Horse immunoglobulins against the autumno-summer tick-borne encephalitis virus could be disintegrated with the aid of papaine to monovalent Fab-fragments which (according to the complement fixation reaction, the test of suppression of the complement fixation, and the HAIT) retained the serological activity whose level was compared with that of the serological activity of gamma-globulin. Fab-fragments possessed a mark...
Poskus E, Zakin MM, Fernámdez HN, Paladini AC.Peptide fragments, obtained from equine growth hormone by cyanogen bromide cleavage and further chemical treatment, were isolated and identified. Their immunological reactivities were tested by hemagglutination and complement fixation methods using rabbit antisera against native hormone. Antigenic determinants were detected in the fragments comprising amino acid sequences 5-72 and 73-123, this last one being predominant. Fragment 124-178 had very low reactivity. Nitration of peptide 73-123 did not modify its immunological properties,but oxidation diminished them. Comparison of the antigenicity...
Watson ED.Uterine washings were found to promote neutrophil mediated killing of Streptococcus zooepidemicus. Depletion of complement and/or specific antibody from the washings significantly reduced bactericidal activity. Phagocytosis of yeast by uterine washings was complement dependent. Inhibition of the classical pathway significantly reduced opsonic activity indicating that, in addition to direct activation via the alternate pathway, antibody may also be involved in yeast phagocytosis.
Boch J.Babesia infections serologically diagnosed in horses, cattle and dogs in Southern Germany during the last few years are described. 321 sera of horses were examined for specific antibodies to Babesia by means of CFT and IIF in 1984; 18 sera reacted to Babesia equi and 4 to Babesia caballi antigen. In a cattle breeding area in the Western Allgäu 13% of 1616 cattle reacted positive to Babesia divergens antigen using IIF and ELISA; during the grazing season 1982 new latent infections were observed in 25 of 266 calves and heifers. Cases of introduced canine babesiosis are more frequent; 10 of 34 s...
Zakin MM, Peña C, Poskus E, Stewart JM, Paladini AC.The immunological properties of a synthetic human growth hormone fragment comprising the amino acids 73 through 128 and of the homologous natural horse growth fragment formed by amino acids 73 through 123, have been comparatively studied. Antisera obtained in rabbits inoculated with the native human hormone or with the fragments, were used. By hemagglutination experiments both fragments have the same reactivity toward the anti-human growth hormone serum, but complement fixation curves detect the existence of at least two populations of antibodies presumably originated against the sequence 73-1...
The Journal of hygieneAugust 1, 1981
Volume 87, Issue 1 93-100 doi: 10.1017/s0022172400069278
Lemcke RM, Ernø H, Gupta U.Two unidentified mycoplasmas, N3 and N11, isolated from the respiratory tract of horses, were found to cross-react with strains of M. mycoides subsp. mycoides in indirect immunofluorescence tests, growth-inhibition tests carried out by the running drop/agar-well method, and in complement-fixation and double immunodiffusion tests. Serologically, the equine mycoplasmas were not completely identical with any of the reference strains of M. mycoides with which they were compared. Their cultural characteristics, ability to digest coagulated serum and casein, and survival at 45 degrees C, however, su...
INGRAM DG.A method is described for the quantitative measurement of the reactions between sensitized cells and horse complement and between alexinated cells and conglutinin. The method is laborious but its application has allowed the determination of the optimal times of the reactions at various temperatures. The results obtained in these experiments indicate that the alexinated configuration with which conglutinin and immuno-conglutinin react is not one of the recognized intermediates formed during the process of immune haemolysis.
Pascoe RR, Bagust TJ.Equine coital exanthema can be produced experimentally in stallions by inoculation with an equine herpesvirus (strain 65/61) and be transmitted during coitus with an infected mare. Serological responses to this infection include the production of complement-fixing and serum-neutralizing antibodies which reach maximum levels 14 to 21 days after infection. Complement-fixing antibodies decline rapidly and are usually not detectable by 60 days after infection, whereas serum-neutralizing antibody activity is maintained for at least 1 year. This disparity provides a useful method for the diagnosis o...
Barron AL, Caste PG, Paul B, Page LA.Postinoculation sera collected from pigeons, turkeys, guinea pigs, sheep, a calf, a rabbit, and a horse experimentally infected with various strains of Chlamydia psittaci yielded a high incidence of positive reactions when tested by double diffusion in gel. Antigen was a deoxycholate extract of SA-2 strain of C. trachomatis. Good correlation was obtained with results of complement fixation tests, whereas double diffusion in gel was less sensitive. Immunoelectrophoresis of the antigen revealed presence of two antigens in the extract.
Anderson LW, Banks KL.Equine macrophages were obtained from female Shetland ponies by injection of Escherichia coli lipopolysaccharide through the lactiferous ducts of the mammary gland. After 6 to 11 days, balanced salt solution was injected into the mammary gland to wash out accumulated cells. Harvested cells contained a mixture of macrophages, lymphocytes, and neutrophils, with the majority of the cells of mononuclear type. In culture, cells adherent after 24 hours were characterized as macrophages by morphologic features, nonspecific esterase staining, and by the presence of complement and immunoglobulin recept...
Boschwitz JS, Timoney JF.A rapid method for purifying equine C3 which yields milligram quantities of pure C3 is described. Protein from equine plasma was selectively precipitated with polyethylene glycol, and the C3 was purified by anionic and cationic exchange HPLC. The yield from this procedure was 12%. The purified C3 was composed of an alpha chain (118 kD) and a beta chain (68 kD) linked by at least one disulfide bond, and it had an isoelectric point of 4.7. Amino acid analysis indicated a strong conservation of amino acid usage between equine and human C3. The N-terminal sequences of the alpha and beta chains wer...
Lavoie JP, Spensley MS, Smith BP, Bowling AT, Morse S.Serum complement activity and selected hematologic variables were evaluated in 5 newborn foals fed bovine colostrum (principal group) and 6 foals allowed to nurse their dam (control group). Also, bovine colostrum was evaluated for anti-equine antibodies. Precolostral serum hemolytic and conglutinating complement activities were low and increased similarly in foals of both groups to reach adult values between 1 and 3 weeks after birth. Bovine colostrum strongly agglutinated, but did not hemolyse principal foals' RBC and blood containing all known equine blood group alloantigens. Hemolysis was n...
McGuire TC.Isolated equine immunoglobulin (Ig)G(T) antibodies to equine infectious anemia virus P26 antigen did not precipitate with antigen when the ratio of antibody to antigen was high. However, at lower ratios of antibody to antigen precipitation occurred. In addition, complement-fixation by IgG and P26 antigen was inhibited by high concentrations of IgG(T). The unusual reaction pattern noted with IgG(T) antibodies was still detectable by the immunodiffusion test for equine infectious anemia virus. In situations of nonprecipitability by IgG(T), the adjacent positive control line was inhibited, and th...
Rashmir-Raven AM, Coyne CP, Fenwick BW, Gaughan EM, Andrews GA, DeBowes RM.The ability of polysulfated glycosaminoglycans (PSGAG) to inhibit the complement cascade was evaluated. The role of complement in inflammation and infection has been well documented. Inhibition of the complement cascade by PSGAG could explain why intra-articularly administered PSGAG diminish diarthrodial joint inflammation and potentiate septic arthritis in horses. Hemolytic complement testing was performed to evaluate the effect of PSGAG on the equine classical and alternate pathways of complement, using rabbit erythrocytes as the target cells. Concentration of PSGAG between 0.2 mg/ml and 0.6...
Baldwin WM, Armstrong LP, Samaniego-Picota M, Rahimi S, Zachary AA, Kasper EK, Conte JV, Hruban RH, Rodriguez ER.Polyclonal antithymocyte globulin preparations contain antibodies with reactivity to endothelial cells. Therefore, we investigated whether treatment with this reagent caused complement deposition in human cardiac transplants. Frozen tissue was available from endomyocardial biopsies of 75 patients, who were transplanted between April 1995 and April 2000. Nine of these patients were converted from cyclosporin A (CsA) to horse antithymocyte globulin (ATGAM) in the first month after transplantation. All of the biopsies were stained by immunofluorescence for C4d as evidence of activation of the cla...
Rehbein G, Heidrich-Joswig S.Eight ponies infected with Babesia equi were investigated for their serological response to B. equi schizont and piroplasm antigen with the indirect fluorescent antibody test (IFAT) and complement fixation test (CFT). Piroplasm antigen was prepared from an infected splenectomized pony, while schizont antigen was produced from cultured lymphoid cells which contained B. equi macroschizonts. The IFAT detected a rise in antibody titres to schizont antigen as well as to piroplasm antigen, but differences were obtained in the duration of antibody detection. Significant antibody titres to piroplasm a...
Rommel FA, Sahu SP.Intrauterine inoculation of pony mares with the bacterium that is the causative agent of contagious equine metritis (CEM) resulted in clinical disease. A humoral immune response could be detected by agglutination and complement fixation (CF), and in some cases precipitating antibody was found by immunodiffusion tests. Agglutinating antibody was the most reliable serological indicator of overt infection and was detected in 8 ot 28 mares after initial intrauterine inoculation of 3-4 x 10(5) bacteria. Seventy percent of mares given a second inoculation and all mares given a third inoculation of 3...
Bannai H, Nemoto M, Tsujimura K, Yamanaka T, Kondo T, Matsumura T.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...
Siegel RC, Cathou RE.Complexes of IgM equine anti-dansyl antibodies and different dansyl substituted carriers were tested for their ability to fix complement (C). Only dansyl92-Ficoll and dansyl12-poly-L-lysine were found to be effective. Dansyl13-bovine serum albumin, dansyl127-keyhole limpet hemocyanin, and reduced and alkylated dansyl10-ribonuclease were all ineffective. Lack of C fixation by the dansyl-ribonuclease was not due to lack of antibody-antigen complex formation, since binding at the concentrations employed for C fixation was established. However, in contrast, polymerized dansyl-ribonuclease (polydis...
Dyer RM, Leid RW.Isolated equine alveolar macrophages obtained by bronchopulmonary lavage of four live ponies demonstrated surface receptors for equine IgG, equine IgM, and complement-coated sheep red blood cells, but not equine IgM or complement-coated erythrocytes alone. In addition, demonstration of IgG receptors was found to depend on the level of erythrocyte sensitization and could not be demonstrated by red blood cell rosetting techniques at low levels of sensitization. Demonstration of receptors for equine complement by red cell rosetting techniques required the presence of both IgM antibody and serum d...