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

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

Comparison of a quantitative microtiter method, a quantitative automated method, and the plate-count method for determining microbial complement resistance.

A quantitative microtiter method for determining the degree of complement resistance or sensitivity of microorganisms is described. The microtiter method is compared with a quantitative automated system and the standard plate-count technique. Data were accumulated from 30 avian Escherichia coli isolates incubated at 35 C with either chicken plasma or heat-inactivated chicken plasma. Analysis of data generated by the automated system and plate-count techniques resulted in a classification of the microorganisms into three groups: those sensitive to the action of complement; those of intermediate sensitivity to the action of complement; and those resistant to the action of complement. Although the three methods studied did not agree absolutely, there were statistically significant correlations among them.     

Diagnostic sensitivity and specificity of an enzyme-linked immunosorbent assay for the diagnosis of Brucella ovis infection in rams.

An enzyme-linked immunosorbent assay (ELISA) for antibody to Brucella ovis was compared with a standard complement fixation test. Sera of 176 rams from uninfected flocks gave 175 negative and one suspect ELISA reaction (diagnostic specificity 99.4%) whereas the complement fixation test yielded 167 negative, seven suspect and two anticomplementary reactions (diagnostic specificity of 96.0%). Diagnostic sensitivity was evaluated on sera of 79 rams from which B. ovis had been isolated. The ELISA showed 75 positive and four suspect reactions, while complement fixation test revealed 64 positive, 13 suspect and two negative results. Considering both positive and suspect reactions, the diagnostic sensitivity was 100% for ELISA and 97.5% for complement fixation test. The ELISA method was considered more specific, more sensitive and technically more advantageous than complement fixation test as a serodiagnostic test for B. ovis infection in rams.     

Complement resistance,as determined by viable count and flow cytometric methods,and its association with the presence of iss and the virulence of avian Escherichia coli

Previous work in our labs has shown that avian Escherichia coli virulence is correlated with resistance to complement. Also, our studies have revealed that the presence of the increased serum survival gene (iss), known to contribute to the complement resistance and virulence of mammalian E. coli, may predict the virulent nature of an avian E. coli isolate. This relationship warrants further research, but further clarification of the relationship among virulence, complement resistance, and iss sequences requires use of complement susceptibility assays. Such assays, unfortunately, are labor-intensive, expensive, and difficult to perform. In the present study, the results of two complement susceptibility assays for 20 E. coli isolates, 10 incriminated in avian colibacillosis and 10 from the intestinal tracts of apparently healthy birds, were compared in an attempt to determine if flow cytometric analysis was a reasonable alternative to a viable count assay. In addition, the virulence of these isolates for chick embryos was determined, and each isolate was examined for the presence of iss using amplification techniques. The flow cytometric method was found to be repeatable for most isolates, and its results showed moderate agreement with those obtained through viable counts. All intestinal isolates of healthy birds proved avirulent using the embryo lethality assay; however, not all isolates from sick birds were demonstrated to be virulent. Possible explanations of these results include that the methods originally used to isolate these organisms failed to detect the illness-inciting strains or that the virulence of these strains had declined following initial isolation. Additionally, we must consider the possibility that the embryo lethality assay of virulence used here might not be sensitive enough to detect differences between these two groups of isolates. Also, it should be noted that virulence assays, such as the one used here, fail to account for predisposing host or environmental conditions, enabling a less virulent isolate to cause disease under natural conditions. Interestingly, the complement resistance of a strain was significantly associated with its lethality in embryos, and iss-containing isolates were significantly more likely than those lacking iss to be classified as complement-resistant and virulent. Such results, at least for this group of avian E. coli, suggest that there is a compelling but imperfect relationship among complement resistance, virulence, and the presence of iss. These results also suggest that the flow cytometric assay may be a reasonable alternative to the viable count method of determining complement resistance.     

A hemolytic assay for the measurement of equine complement

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

Comparison of the enzyme-linked immunosorbent assay and the indirect hemagglutination and complement fixation tests for detecting antibodies to Mycoplasma hyopneumoniae.

Caesarean-derived, colostrum-deprived swine were exposed to a broth culture of a low passage field isolate of Mycoplasma hyopneumoniae by intranasal inoculation. The intranasal-inoculated swine subsequently were commingled with their litter-mates to effect transmission via contact-exposure. Sera were collected from the swine at two to four week intervals for approximately one year postexposure and evaluated by the enzyme-linked immunosorbent assay (ELISA), indirect hemagglutination and complement fixation tests. The intranasal-exposed swine seroconverted earlier, developed higher titers and remained indirect hemagglutination and complement fixation positive longer than the contact-exposed swine. It was concluded that the antibody response of intranasal-exposed swine was artificially high and that sera from such swine were not suitable for evaluating the sensitivity of mycoplasmal pneumonia of swine serodiagnostic tests. The indirect hemagglutination test was relatively insensitive and technically cumbersome and the least promising as a practical field test. The complement fixation test appeared to be slightly more sensitive in detecting early antibody production (especially in contact-exposed swine) but it was the least sensitive in detecting late antibodies. The ELISA was generally the most sensitive procedure. Individual high ELISA titers were from ten to 32 times greater than maximum complement fixation and indirect hemagglutination titers. The most striking difference among the three tests was the persistence of high ELISA titers late in the study. All swine were ELISA positive at necropsy approximately one year postexposure despite the fact that lungs were devoid of lesions and culturally and immunofluorescent negative for M. hyopneumoniae.     

Myopathy of dogs resembling white muscle disease of sheep

Automation of the complement fixation test for diagnosing Brucella abortus infection in cattle has allowed this difficult, time-consuming and labour-intensive hut most specific test to he used for large scale serodiagnosis in New Zealand's Brucellosis Eradication Scheme. The automated test has the advantages of eliminating much human error, of improving accuracy and of reducing the costs of labour and reagents.

The Auto-Analyzer performs a warm complement fixation test at a single serum dilution. Each batch of 39 serum samples is compared with a standard positive control serum with a complement fixing antibody activity equivalent to 1/30 of the second International Standard Ani-Brucella abortus Serum. All sera with an antibody level equal to, or greater than, the standard serum are regarded as positive to the test. A titre for serum tested by the automated method is not obtained, and positive sera that prozone strongly may be recorded as negative. Where necessary, the semi-automated microtitre complement fixation test is used to delineate titres and the status of doubtful automated CFT results. The automated and microtitre complement fixation tests use the same reagents; only relative concentrations differ.     

Comparison of the enzyme-linked immunosorbent assay and complement fixation test for detecting Brucella ovis antibodies in sheep

A solid-phase, indirect, enzyme-linked immunosorbent assay (ELISA) was compared with the microtitre complement fixation test for detecting Brucella ovis antibodies in 220 ram sera. The ELISA was more sensitive than the complement fixation test; it demonstrated antibodies in 11 sera from known infected or vaccinated rams that were complement fixation test negative. No false positives were recorded with the ELISA and, in 36 sera positive to both tests, the ELISA titres were consistently higher than the corresponding complement fixation test titres.     

A single dilution enzyme-linked immunosorbent assay for the quantitative detection of antibodies to African horsesickness virus

A single dilution enzyme-linked immunosorbent assay (ELISA), utilizing a reference serum with a known, predetermined end-titre was used to compute antibody titres of serum samples against African horsesickness virus. Compared to the complement fixation test it was found to be highly reproducible and sensitive. The assay also reduced the effect of between-test variations on test results and proved superior to an existing single-dilution ELISA method.     

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.     

Comparative evaluation of enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of dourine

The detection of antibodies against Trypanosoma equiperdum in 689 equid sera was compared by enzyme-linked immunosorbent assay (ELISA), the complement fixation test (CFT) and an indirect immunofluorescent test (IIF). CFT was the least sensitive technique, susceptible to anti-complementary factors and the most technically demanding. IIF was more sensitive, but was only suitable for testing limited numbers of samples. In this study, ELISA was the most sensitive test, the least labour intensive and lends itself to a considerable degree of automation. It is suggested that ELISA would be relatively easy to standardise between laboratories and an ELISA protocol could be adopted as the internationally approved test for equine health certification.     

Comparison of serological tests for the detection of antibody to natural and experimental murine cytomegalovirus.

Three serological tests, i.e. complement fixation test, indirect immunofluorescent assay, and enzyme-linked immunosorbent assay (ELISA) were compared for sensitivity in the detection and titration of murine cytomegalovirus antibody. The three tests were compared using sera from experimentally inoculated and naturally infected mice bled at intervals from 3 to 140 days postinfection. In the acute infection, complement fixation and indirect immunofluorescent assay tests were of comparable sensitivity for early detection of antibody, whereas the ELISA was less sensitive. In persistent infection, higher titers were recorded with ELISA. Since murine cytomegalovirus has been shown to exert significant effects on the immune response of infected mice, this antigen should be included routinely in viral antibody screening programs.     

Antibody and complement mediated lysis of felid herpesvirus 1 infected cells in vitro

The lysis of cells infected by felid herpesvirus 1 (FHV) by feline anti-FHV antibody and complement was demonstrated. Lytic activity was sensitive to dilution of both antibody and, especially, complement. It was first detected within 10 to 20 minutes, increased rapidly during the next 30 minutes of incubation and then rose more slowly in a linear manner. Using standard antibody and complement concentrations and assay duration, it was shown that FHV infected cells underwent significant (P less than 0.05) lysis from eight hours after infection in a system in which FHV-specific membrane antigen was first detected at three hours after infection and spread of FHV by the intracellular route began eight to nine hours after infection. The ability of antibody and complement to reduce FHV spread in this system was demonstrated by a significant (P less than 0.05) reduction in FHV plaque numbers, although the restriction of spread was not absolute. Chelation of divalent cations and heat inactivation of complement factor B revealed that the lytic system was dependent on factor B and Mg2+ but not Ca2+, suggesting involvement of the alternative pathway of complement activation.     

Detection and typing of foot-and-mouth disease virus by enzyme-linked immunosorbent assay: a sensitive, rapid and reliable technique for primary diagnosis

A highly sensitive indirect sandwich enzyme-linked immunosorbent assay suitable for adoption as the routine diagnostic and typing test for foot-and-mouth disease virus of all seven serotypes is described. The assay uses rabbit and guinea pig antisera raised against inactivated 146S virus antigens. Strong homotypic and minimal heterotypic reactions with both whole virion 146S and derived virion subunit 12S antigens achieved a detection sensitivity approximately 125 times that of the complement fixation test. When applied to diagnostic material with positive-negative threshold criteria generated from testing a large number of negative samples, a positive result was obtained on 83.3 per cent of original virus-positive epithelia, three times the rate for the complement fixation test, and all were typed after one passage in culture.     

An investigation into alternative methods for the serodiagnosis of dourine

The complement fixation test (CFT), indirect fluorescent antibody test (IFAT), card agglutination test for trypanosomiasis (CATT) and enzyme-linked immunosorbent assay (ELISA) were compared in their application to the serological diagnosis of Trypanosoma equiperdum infection in 43 horses. The CFT remains a reliable test for dourine, especially in countries where other members of the subgenus Trypanozoon do not occur. The IFAT is a good 'back-up' test, but, requiring skilled operators it has the disadvantage of making it labour intensive, and interpretation of results subjective. This makes it more suited to small numbers of samples. The ELISA is suitable for large numbers of samples and could readily be used in routine diagnostic procedures. The CATT could be of value in field situations, although it does not appear to be as sensitive as the CFT. Its possible application under these conditions should be further investigated.     

Contagious caprine pleuropneumonia: Some observations in a field vaccination trial using inactivatedMycoplasma strain F38

The efficacy of an inactivated Mycoplasma strain F38-saponin vaccine in natural infection with contagious caprine pleuropneumonia was investigated. A total of 10,000 goats were vaccinated, out of which 400 were regularly monitored for a period of six months post-vaccination. Immunised animals remained free from infection throughout the period of observation. The antibody response was followed using complement fixation and slide agglutination tests. Both tests could detect F38 antibody in the majority of vaccinated goats but the slide agglutination test was found to be more sensitive than complement fixation. The significance of the results is discussed.     

The effect of removal of adherent cells in lectin and allogeneic cell stimulation of bovine lymphocytes

Macrophages have been shown to play an important role in lymphocyte activation in many species but not unequivocally for bovine. In this study we have shown that when sufficient care is taken to remove macrophages by adherence or by anti-macrophage serum and complement, bovine lymphocytes respond poorly to lectins or to allogenic cells in mixed lymphocyte culture. Addition of macrophages to approximately the same level as found in the unseparated cells restores the proliferative responses. The degree of response shown by the macrophage depleted cultures depends on the lectin concentration. Stimulation with lower doses of lectin is more sensitive to macrophage concentration. The mixed lymphocyte response and the response to Concanavalin A (ConA) are more sensitive to macrophages than is the response to phytohemagglutinin (PHA).     

Syncytium-induction inhibition test with complement for detection of antibodies against bovine leukemia virus.

A modified syncytium-induction inhibition test which is more sensitive than the immunodiffusion test, was developed using rabbit complement. In this test, fetal lamb kidney cells continuously infected with bovine leukemia virus were used as effector cells, and the CC81 cat cells transformed with murine sarcoma virus, were used as indicator cells. The syncytium-induction inhibition effect of anti-bovine leukemia virus serum was enhanced significantly by the addition of rabbit complement. The syncytium-induction inhibition titers had a statistically significant correlation with the immunodiffusion titers and were four to 64 times higher than immunodiffusion titers. In 12 experimentally infected cattle, the syncytium-induction inhibition test detected the antibodies earlier than the immunodiffusion test and continuously detected them when immunodiffusion antibody changed to negative. In the 81 sera from naturally infected herds, 35 (43.2%) were positive by the immunodiffusion test and 55 (67.9%) by the syncytium-induction inhibition test.     

Bovine rotavirus diagnosis: comparison of various antigens and serological tests

Agar gel immunodiffusion (AGID) and counter-immunoelectrophoresis (CIEP), complement fixation (CF), radio-immunoassay (RIA), haemagglutination (HA) and haemagglutination inhibition (HI) tests were compared in their efficiency for the detection of bovine rotavirus antigens and antibodies. As a test for antigen using hyperimmune serum, CIEP was found to have advantages over AGID by being more rapid as well as approximately four times more sensitive regardless of whether the antigen was of faecal or tissue culture origin. The CF test was more sensitive than either of the immunodiffusion procedures studied for antigen detection, but was more tedious to perform and of limited use as some faecal samples exhibited anti-complementary activity. For measurement of rotavirus antibody the radio-immunoassay (RIA) was the most sensitive technique and the CIEP least sensitive. Using the RIA a limited survey of cattle demonstrated that approximately 75% of the animals tested possessed specific antibody to rotavirus.