Hemagglutination Inhibition with Arboviruses: Relationship Between Titers and Source of Erythrocytes

Antigens for Grand Arbaud, Hazara, and California arboviruses were able to agglutinate goose and either dog, hamster and guinea pig, or hamster red blood cells (RBC) to the same titer at the same pH; in hemagglutination-inhibition (HI) tests, titers for homologous and related sera were the same with these different types of RBC or occasionally one dilution higher with the mammalian cells. Antigens for St. Louis encephalitis and Eastern equine encephalitis viruses required use of lower antigen dilutions with human, guinea pig, and hamster RBC than with goose RBC. The results of comparative HI testing with these latter antigens and types of RBC indicate that HI titer is not directly related to the antigen dilution used with different types of RBC.     

Specificity and Sensitivity of the Streptozyme Test for the Detection of Streptococcal Antibodies

A comparison between the results of the streptozyme hemagglutination test and serological titers for anti-streptolysin O (ASO), anti-hyaluronidase (AH), anti-deoxyribonuclease B (ADN-B), and anti-nicotinamide adenine dinucleotidase (ANAD) was made in two groups of human sera. In one group, serological titers for all the four antibodies were lower than the threshold of sensitization reported by the producing firm. In the second group, the titer of at least one of the four antibodies was equal to or higher than the threshold. False-positive and false-negative reactions occur with those sera when one or more antibody titer is at or near the threshold of the test as described by the manufacturer. The test was positive for all sera where either the ASO was greater than 166 or the ANAD was greater than 270, and for 98% of the sera with ADN-B greater than 360. It is, therefore, concluded that the streptozyme test can be used as an adjunct to the clinical diagnosis of streptococcal infections and their nonsuppurative sequelae. It is less useful to assess the levels of antibodies in sera from general population surveys. For such sera, the relative specificity and sensitivity of the test might yield misleading results. Until more experience is gained with the test, caution should be used in its application to infant and older adult age groups, where significant streptococcal antibody titers are frequently near the threshold of the test.     

A single serum dilution method for the quantitation of neutralizing antibodies to varicella-zoster virus

A one-point serum dilution method for determination of neutralizing antibody in human sera to varicella-zoster (V-Z) virus instead of the serial serum dilution method was investigated. Focus counting was performed under a microscope on day 5 to 6 after inoculation of V-Z virus into 6-well plastic trays in which human embryonic lung cells were grown. A table was constructed to estimate the ND50 titers by the per cent reduction of the focus count from the control at only one dilution of test sera. The estimated ND50 values agreed well with those determined by the serial serum dilution method. Test sera showed a slight nonspecific reactivity at low serum dilutions, but reliable results could usually be obtained at a serum dilution of 1:8 or more. This method, which saves materials and labor, was applied to the quantification of neutralizing antibody against V-Z virus in human sera with satisfactory accuracy and reproducibility.     

Comparison of Antistreptolysin O Latex Screening Test with the Antistreptolysin O Hemolytic Test

This report describes a comparison of the Behringwerke antistreptolysin O (ASO) latex screening test with the ASO hemolytic test. Agreement between the two tests was poor when Difco streptolysin O (SLO) reagent was employed in the hemolytic test; approximately 34% of the sera with ASO titers in the normal range of the hemolytic test gave false-positive latex test reactions. However, the percentage of false-positive latex test reactions was only 5% when Behringwerke SLO reagent was used in the hemolytic test. An assay of the Difco and Behringwerke SLO reagents against an ASO standard indicated that the Difco SLO reagent was more potent than the Behringwerke SLO reagent. The lack of agreement between the Behringwerke latex test and the hemolytic test using Difco SLO reagent is attributed to the potency of the SLO reagents.     

Guinea pig complement potently measures vibriocidal activity of human antibodies in response to cholera vaccines

The vibriocidal assay using guinea pig complement is widely used for the evaluation of immune responses to cholera vaccines in human clinical trials. However, it is unclear why guinea pig complement has been used over human complement in the measurement of vibriocidal activity of human sera and there have not been comparison studies for the use of guinea pig complement over those from other species. Therefore, we comparatively investigated the effects of complements derived from human, guinea pig, rabbit, and sheep on vibriocidal activity. Complements from guinea pig, rabbit, and human showed concentration-dependent vibriocidal activity in the presence of quality control serum antibodies. Of these complements, guinea pig complement was the most sensitive and effective over a wide concentration range. When the vibriocidal activity of complements was measured in the absence of serum antibodies, human, sheep, and guinea pig complements showed vibriocidal activity up to 40-fold, 20-fold, and 1-fold dilution, respectively. For human pre- and post-vaccination sera, the most potent vibriocidal activity was observed when guinea pig complement was used. In addition, the highest fold-increases between pre- and post- vaccinated sera were obtained with guinea pig complement. Furthermore, human complement contained a higher amount of V. cholerae- and its lipopolysaccharide-specific antibodies than guinea pig complement. Collectively, these results suggest that guinea pig complements are suitable for vibriocidal assays due to their high sensitivity and effectiveness to human sera.     

Comparative studies of the protein composition of red blood cell membranes from eight mammalian species

The polypeptide pattern of red blood cell (RBC) membranes from cow, sheep, horse, rabbit, guinea pig, rat, mouse, analyzed by polyacrylamide gel electrophoresis, was compared to human RBC counterpart. Some qualitative and quantitative differences were noted. Among the high molecular weight components the bands 2.1- 2.3 appeared slightly decreased in rabbit and rat and increased in sheep RBC membranes. Band 3 appeared to have a higher molecular weight in the cow, guinea pig and mouse RBCs, and a lower molecular weight in the sheep RBCs. Band 4.1 from the RBC membranes of cow, sheep, rabbit and guinea pig was splitted into two sub-bands, while band 4.2 overlapped with band 4.1 in horse and guinea pig RBC membranes. There are marked differences in the number and position of bands in the 4.5 region, while band 4.9 is present in higher amounts in horse, rabbit and guinea pig RBC membranes. Band 6 (glyceraldehyde 3-phosphate dehydrogenase) was undetectable in horse, rat and mouse RBC membranes and was decreased in sheep, rabbit and guinea pig. There are also major differences in the region of band 7 and below ("post-7"). Band 8 was undetectable in horse, cow and guinea pig, and was in higher amounts in rat. A band corresponding to a molecular weight of about 22 kD in the "post-8" region was present only in guinea pig RBC membranes.     

Micro Test for Streptococcal Anti-deoxyribonuclease B

A methyl green decolorization micro test for the determination of streptococcal anti-deoxyribonuclease B titers in serum is described. The micro test was compared with an alcohol precipitation macro test on 112 human sera. The anti-deoxyribonuclease B titers agreed within one dilution step for 85.7% of the 112 sera examined by both techniques. Both tests were about equal in the detection of titers above the upper limits of normal. The micro test requires smaller amounts of reagents and fewer man-hours to perform than the macro test. This translates into lower cost per test and more tests per man-hour.     

Specificity and inhibitory activity of antibodies to Plasmodium falciparum aldolase

The multiplication of Plasmodium falciparum within RBC is energy-dependent and the glucose consumption of infected RBC is increased more than 50 times over the consumption of normal RBC. High levels of glycolytic enzymes such as fructose-1,6-diphosphate aldolase (p41) have been detected in infected RBC. Expression of the cloned aldolase gene of P. falciparum in Escherichia coli resulted in an enzymatically active polypeptide with a high sp. act. and the recombinant p41 aldolase was used for enzymatic and immunologic studies reported here. The presence of antibodies against p41 in the sera of human adults partially immune to malaria and immunization experiments in monkeys suggest that p41 is implicated in protective immune response against the parasite. Therefore, we analyzed the capacity of various antisera to inhibit P. falciparum aldolase activity. It was found that anti-p41 antibodies raised in mice, rabbits, and monkeys inhibited very efficiently aldolase activity in vitro up to dilutions higher than 10(-3). In contrast none of the human sera with high levels of anti-p41 antibodies were able to inhibit parasite aldolase activity even at a dilution of 1/2. The inability of human antisera to neutralize parasite aldolase is not related to antibody titers but is probably related to the specificity of the human antibodies. This finding is discussed in relation to homology of structure of P. falciparum and mammalian aldolase and to a possible mechanism of parasite adaptation and survival in its natural host.     

荷斯坦种公牛血清替代补体溶血活性研究

本文将国外脊椎动物血清补体溶血活性标准测定方法,运用到荷斯坦种公牛研究中,首次建立了测定荷斯坦种公牛血清补体溶血ACH50的方法。种公牛血清经相应靶红细胞吸附后,可溶解悬浮在EGTAMgGVB缓冲液中的正常的兔血红细胞、人A,B,AB,O型红细胞,小鼠、大鼠、鸡红细胞,但对绵羊、山羊、猪红细胞溶血活性较低;对奶牛红细胞无溶血活性。且发现种公牛血清的溶血活性和靶红细胞的动物种类在系统发育上和种公牛的亲缘关系远近没有直接联系。种公牛血清在EGTAMgGVB缓冲液中对兔血红细胞发生溶血的最适条件是:温度是37℃,最适pH是7.3-7.4,最适Mg2 的浓度是4mmol/L,最适孵育时间为90min。溶血活性是二价离子依赖、热敏感(溶血活性热灭活温度是56℃)。种公牛血清对兔血红细胞的溶血活性在受到酵母聚糖、甲胺、肼、EDTA、鸡抗酵母聚糖牛血清结合物抗血清处理时,溶血活性可全部或部分消失,溶血活性抑制程度与补体抑制剂浓度相关。我们运用建立的标准溶血方法并以兔血红细胞作为指示细胞检测不同年龄的53头种公牛血清补体替代途径的溶血活性,溶血值在13.2-44.3u/ml之间,还发现不同年龄组公牛之间溶血活性有随年龄增加而逐步增大趋势,但差异不显著(P>0.05),在4-5岁公牛群中达到最大值。对种公牛血清补体系统溶血水平进行系统研究,一方面可以填补国内在此领域研究空白,另一方面也利于种公牛疾病监测、控制,此外也为兽医临床诊断试剂的研制提供新的技术手段。     

Immunologic responses of domestic and bighorn sheep to a multivalent Pasteurella haemolytica vaccine

The efficacy of a Pasteurella haemolytica vaccine (serotypes A1, A2, and T10) to induce humoral antibodies and alter colonization of the upper respiratory tract by related P. haemolytica spp. strains was evaluated in 10 bighorn (Ovis canadensis canadensis) and 10 domestic (Ovis aries) sheep. Sheep of each species were divided into five pairs based on age and history of respiratory disease. One sheep in each pair was vaccinated twice 2 wk apart with 2 ml of vaccine (VAC group) and the remaining animals (NV group) were injected with 2 ml of sterile saline. Mild, transient lameness was the only observed adverse effect. Blood sera from the sheep were tested for agglutinating antibodies against whole cells of A1, A2, and T10 and for leukotoxin neutralizing antibodies. Antibody titers were expressed as the reciprocal log2 of the highest reactive dilutions. Domestic sheep > 1-yr-old and two bighorn sheep with a history of A1 infection had higher titers throughout the study against A1 cells than domestic sheep < 1-yr-old and bighorns without a history of A1 infection. Both domestic and bighorn sheep had log2 titers of 8 to 12 against A2 cells and 6 to 12 against T10 cells during this time. Bighorn sheep in the VAC group had 2 to 32 fold titer increases for A1 cells by 2 wk post-vaccination (PV) compared to 0 to 2 fold increases in VAC domestic sheep. Two to 16 and 0 to 8 fold increases in antibodies titers to A2 and T10 cells, respectively, were detected in sera of both VAC groups. Sera of bighorn sheep with a history of respiratory disease and all domestic sheep had log2 leukotoxin neutralizing antibody titers of 4 to 14 in contrast to < or = 2 in sera of bighorn sheep without a history of respiratory disease. Neutralizing antibody titers of two bighorns without a history of respiratory disease in the VAC group increased from log2 0 to 5 in one and from 0 to 9 in the other 2 wk PV. Antibody increases in these animals were no longer evident at 16 wk PV while titers of animals with histories of disease remained relatively stable. The types and numbers of Pasteurella spp. isolated from nasal and pharyngeal swabs varied throughout the study without conclusive evidence of suppression of colonization. Although the animals were not experimentally challenged to determine the efficacy of the vaccine, one VAC and one NV bighorn sheep died following introduction of an A2 P. haemolytica strain when leukotoxin neutralizing antibodies had returned to pre-vaccination levels. This vaccine appeared to be safe for use in bighorn sheep and stimulated moderate but transient increases in antibody levels which should provide some protection against naturally occurring disease. A vaccine which would induce production of high and maintained antibodies against multiple strains of P. haemolytica would be valuable for use in bighorn sheep maintained in captivity or when captured for relocation.     

Occurrence of hemolytic activity in the skin secretion of the caecilian siphonops paulensis.

The skin secretion of the caecilian Siphonops paulensis (SpSS) induces a time-and dose-dependent hemolytic response on red blood cells (RBC). When RBC from various animals species were subjected to the action of SpSS, a range of sensitivities was evident, sheep erythrocytes being the most susceptible, human, mouse and rabbit having moderate susceptibility, cow, snake and toad erythrocytes being more resistant, while S. paulensis RBC were entirely resistant. The hemolytic activity of SpSS was inhibited at temperatures higher than 60 degrees C. Both trypsin- and chymotrypsin-treated SpSS were ineffective in inducing RBC lysis. The treatment of SpSS with sheep RBC ghosts reduced its activity. There is no phospholipase activity in the SpSS.     

The application of different cell lines and virus strains for detection of immunofluorescence rubella antibodies.

Fluorescence antibodies (FA) were titrated in human sera against antigens synthesized in four different infected cell lines: SIRC, RK-13, Vero and BHK-21. The cells infected in suspension by rubella virus attained the optimal concentration of fluorescence staining antigen earlier, fluorescence was more intensive and the titers of the tested human sera were slightly higher than in monolayer cell cultures. FA titers were high and they were correlated with the antibody titers obtained by hemagglutination-inhibition test. Some practical implications of these findings are discussed.     

AN INDIRECT ENZYME-LINKED IMMUNOSORBENT ASSAY FOR THE DETECTION OF LEPTOSPIRA INTERROGANS SEROVAR POMONA ANTIBODIES IN BOVINE SERA

An indirect ELISA was developed and initially evaluated for the detection of bovine antibodies to Leptospira interrogans serovar pomona. The antigen used in this ELISA was extracted from a serovar pomona culture supernatant by a combination of centrifugation, digestion with proteinase K and ultra-centrifugation. The antigen showed little cross-reaction with immune rabbit sera to L. interrogans serovars copenhageni, grippotyphosa, hardjo and sejroe and, Leptospira biflexa serovar patoc. Some cross-reaction was observed with immune rabbit serum to L. interrogans serovar canicola. The relative sensitivity of the ELISA was 94.76% confidence interval =± 3.32%) when estimated with bovine sera (n=172) with serovar pomona microscopic agglutination test (MAT) titers of 100. The relative specificity of the ELISA was 99.28% (95% confidence interval = 1.40%) when estimated with bovine sera (n=139) with MAT titers of <100 to L. interrogans serovars canicola, copenhageni, grippotyphosa, hardjo, pomona and sejroe. Thirty six of 258 field sera (13.95%) with serovar pomona MAT titers of <100, gave positive reactions in the ELISA.     

Chromium-51 labeling of sheep red blood cells.

The failure of sheep red blood cells (RBCs) labeled with Chromium-51 (Cr-51) using the ascorbic acid technique to act as a suitable intravascular marker of blood volume in a septic sheep model prompted us to investigate the technique of radiolabeling sheep erythrocytes with this isotope. Consequently, we studied thirteen sheep in which the labeling efficiency of Cr-51 as sodium chromate and hemoglobin typing was determined for each animal. Mean Cr-51 labeling efficiency of sheep RBCs was 67.5% (n = 13). Although 5 of the 13 sheep were discovered to have two types of hemoglobin (Hb) as determined by electrophoresis, overall labeling efficiency of sheep RBCs was determined to be independent of the type of hemoglobin present. However, when two types of Hb were present (Hb-A and Hb-B), Cr-51 had a higher affinity for Hb-B (80%) than Hb-A (20%) even though both Hb types are present in similar proportions (Hb-A = 53%, Hb-B = 46%). The results of this study indicate that sheep RBCs express a lower labeling efficiency for Cr-51 than do human RBCs and that Cr-51 has a higher affinity for Hb-B than for Hb-A when both hemoglobin types are present. This difference is noteworthy when interpreting Cr-51 RBC data in experimental sheep models. Furthermore, caution should be exercised when extrapolating established human protocols to animal models.     

红斑性肢痛症相关痘病毒血清免疫学特征的研究

本文从四个方面综合报道了红斑性肢痛症相关痘病毒(ERPV)血清免疫学特征的研究结果。血清学鉴定表明,ERPV为正痘病毒属内成员,但与正痘病毒属内成员痘苗病毒和鼠痘病毒具有中和抗原表位差异。流行性红斑性肢痛症患者血清中含有抗ERPV的中和抗体和抗ERPVA型包涵体IgG抗体,其检出率明显高于当地未发病者和美国人群的检出率。     

Detection of Coronavirus 229E Antibody by Indirect Hemagglutination

Tannic-acid treated sheep erythrocytes (fresh or glutaraldehyde preserved) were sensitized with 229E antigens from human embryonic lung (RU-1) cell cultures. Indirect hemagglutination (IHA) antigen titers in 229E-infected cell cultures paralleled virus infectivity and complement fixation (CF) antigen titers. The identity of the IHA antigen was confirmed by testing extracts from inoculated and control cell cultures for ability to inhibit IHA. Also, significant increases in IHA antibody were demonstrated with acute and convalescent serum pairs from patients with proven 229E infections. A comparison of IHA, neutralization and CF titers for 229E antibodies was made on human sera drawn from different populations. The IHA and neutralization results were in agreement on 93% of the 129 sera found to be positive by at least one of three tests. The number of antibody titers detected by the CF test was insufficient to permit comparison. Hyperimmune sera from animals immunized with OC 43 did not react with 229E by IHA. Also no increase in IHA antibody was demonstrated with acute and convalescent serum pairs from patients with seroconversions to OC 43. These findings suggest that the IHA test provides (i) a rapid and sensitive method for serodiagnosis of 229E infections and (ii) a simple and inexpensive method for seroepidemiological studies.     

Antistreptolysin O titer in horses

Antistreptolysin O (ASO) titers were determined in the sera of 532 horses from stud farms and 436 working horses from small farms. A statistically significant correlation was seen between the ASO titer and the age of the horses. There was a significant difference between mean ASO titer in horses 0-2 years and horses 2-10 years In horses older than 10 years the titer was significantly higher than mean ASO titer for the group. Twenty four of 30 horses in which ASO titer was higher or equal to 80 I.U. had histories which suggested that streptococcal infection had occurred. Clinical and bacteriological examination showed that in all examined stud farms cases of Streptococcus C infection was noted during the 2 years before serum was collected. Streptococcus C was isolated usually from horses which had low ASO titer. Our results are in agreement with the suggestion that S. zooepidemicus infections increase the ASO titer in serum of horses.     

A comparative study of animal erythrocyte agglutinins from marine algae

1. Fifteen marine algal species were analyzed for agglutinins to rabbit, sheep and human A, B and O blood group erythrocytes. 2. Protein extracts from all marine algae agglutinated rabbit erythrocytes, whereas twelve and five extracts agglutinated sheep and human erythrocytes, respectively. 3. The highest agglutination titers were consistently observed with rabbit erythrocytes. 4. Dictyota dichotoma strongly agglutinated human B blood group erythrocytes relative to A and O group erythrocytes. 5. Agglutination titer of rabbit erythrocytes by six algal extracts was not inhibited by mono- or polysaccharides, yet was reduced by glycoproteins.     

Effect of animal sera on Bacillus anthracis Sterne spore germination and vegetative cell growth

Aims: The aims of this work were to investigate the effects of sera on B. anthracis Sterne germination and growth. Sera examined included human, monkey and rabbit sera, as well as sera from eight other species. Methods and Results: Standard dilution plate assay (with and without heat kill) was used as a measure of germination, and spectroscopy was used to measure growth. In addition, a Coulter Counter particle counter was used to monitor germination and growth based on bacterial size. Spores germinated best in foetal bovine and monkey sera, moderately with human sera and showed limited germination in the presence of rabbit or rat sera. Vegetative bacteria grew best in foetal bovine sera and moderately in rabbit sera. Human and monkey sera supported little growth of vegetative bacteria. Conclusion: The data suggested sera can have a significant impact on germination and growth of Sterne bacteria. Significance and Impact of the Study: These data should be considered when conducting in vitro cell culture studies and may aid in interpreting in vivo infection studies.     

Erythrocytes of Japanese Quail for Hemagglutination by Rubella Virus

Erythrocytes (RBC) of adult Japanese quail, Coturnix coturnix japonica, were found to be as sensitive as day-old chick RBC for rubella virus hemagglutination (HA). Various factors involved in the HA were studied with quail RBC as well as with adult pigeon and day-old chick RBC. Pigeon RBC, unlike chick and quail RBC, tended to show, without hemagglutinin, a pattern of sedimented RBC resembling the HA pattern by the virus at 4 C, to a lesser extent at room temperature, in 0.85% NaCl solution buffered with m/100 phosphate (PBS), and were prevented from doing so by addition of a small amount of bovine plasma albumin to the diluent. A small amount (about 10^?3 m ) of CaCL₂ in PBS gave higher HA titers. The HA titer was higher at 4 C than at room temperature and much reduced at 36 C with chick and quail RBC. With pigeon RBC, addition of bovine plasma albumin to the diluent tended to reduce HA titers. The HA titer was highest at pH 5.8 to 6.8, and was inversely proportional to the RBC concentration. Under the conditions established on the basis of these findings, chick and quail RBC gave similar HA titers, but pigeon RBC gave consistently higher titers. However, these types of RBC gave no significant difference in HA-inhibiting antibody titer when 4 units of hemagglutinin as determined with the homologous RBC was used.