Immunopotentiation of outer membrane protein through anti-idiotype Pasteurella multocida vaccine in rabbits

Pasteurella multocida was isolated from cattle affected with haemorrhagic septicaemia and characterized on the basis of morphological, cultural and biochemical tests. Bacterial outer membrane proteins (OMPs) were extracted with 1% Sarkosyl method. P. multocida anti-idiotype vaccine prepared from OMPs (21.3 mg per 100 ml), was evaluated and compared with bacterin supplemented with 10% OMPs and plain alum-adsorbed bacterin in rabbit models. It was observed that OMPs-anti-idiotype vaccine induced high levels of antibody titres (geomean titres -GMT) detected using indirect haemagglutination (IHA) test. The OMPs anti-idiotype antibody titres of 168.9 GMT were obtained to 42.2 GMT in OMPs supplemented bacterin on 21 days post vaccination, while the plain bacterin had the least titre of 27.9 GMT. The OMPs-anti-idiotype vaccine provoked better immunogenic response in terms of highest GMT titres and long lasting effect in rabbits and 100% protection against the challenge with homologous strain of P. multocida,while 88% protection was obtained in rabbits, given OMPs supplemented bacterin.     

Studies on haemagglutination by the EMC-MM-Columbia SK-group of viruses

Summary When MM and Columbia SK virus were propagated in the chick embryo, the virus disappeared from the allantoic fluid and from the embryonic brain tissue after 6 to 9 chorioallantoic passages. Amniotic passages, however, resulted in a gradual increase of the mouse infectivity titre of the amniotic fluid and of the fluid of the regularly observed subcutaneous edema of the embryos, though after 22 passages the ID50 titre was still 3 logs lower than that of the original mouse brain suspension. The virus was present not only in amniotic fluid, but also in allantoic fluid and throughout the whole embryo. In spite of the pretty high mouse infectivity titres, the extra-embryonic fluids failed to produce haemagglutination of sheep red cells, whereas low haemagglutination titres could be obtained with suspensions of embryonic brain tissue. No evidence was obtained, that a non-specific inhibitor was responsible for the inability to produce haemagglutination. The virus present in egg fluids is only partially adsorbed, which is in contrast with the almost complete adsorption of virus in mouse brain suspension. The adsorption of a relatively small amount of virus might be one of the factors responsible for lack of haemagglutinating ability. It is suggested, that another coinciding factor, which is necessary for the initiation of the haemagglutination might be present in the central nervous system lesion.Third publication: Antonie van Leeuwenhoek17, 137, 1951.     

Anti-A and anti-B haemagglutinin levels in intravenous immunoglobulins: Are they on the rise? A comparison of four different analysis methods and six products

Recent reports of severe haemolytic reactions upon high dose treatment with new generation intravenous immunoglobulins (IVIGs) prompted us to examine the anti-A and anti-B haemagglutinin content of these therapeutics. We compared four different test methods, namely the indirect and direct haemagglutination test as described in the European Pharmacopoiea (Ph. Eur.) and two commercial gelcard systems with the aim to define the most reliable method for a large-scale comparison of different IVIG products. Absolute titres varied when the same samples were analyzed by the four methods, while the relative ranking of six different IVIG preparations representing different manufacturing classes was identical. New generation IVIGs showed 1–2 titre steps higher anti-A titres than the older products. Haemagglutinin titres of all 48 IVIG batches analyzed were within the current Ph. Eur. specification of ≤1:64 when tested by the official pharmacopoeial method. Based on efficiency, reliability and lower costs, the direct gelcard method could be a valid alternative to the official Ph. Eur. method to serve as a limit test. However, due to the highest intermediate precision, the official Ph. Eur. method seems to be most suitable to compare haemagglutinin titres of different IVIG products.     

Xylanase production by Aspergillus awamori. Development of a medium and optimization of the fermentation parameters for the production of extracellular xylanase and beta-xylosidase while maintaining low protease production

A growth medium was developed for maximal production in batch culture of extracellular xylanase and beta-xylosidase by Aspergillus awamori CMI 142717 and a mutant (AANTG 43) derived from the wild-type strain. The optimum pH for the production of xylanase and beta-xylosidase was 4.0. The best temperature of xylanase production was 30 degrees C; 35 degrees C was optimal for beta-xylosidase. Protease production was never completely suppressed under any of the conditions tested. However, protease titre was 3.5-fold less than the control in medium in which proteose peptone and yeast extract were omitted: the level of xylanase was not affected (8.6 U mL(-1)) but beta-xylosidase titre was increased 4.7-fold to 1.5 U mL(-1). When corn steep liquor was used as the sole nitrogen source, xylanse and beta-xylosidase titres were further increased by 1.5- and 1.9-fold, respectively. Of the carbon sources investigated, ball-milled oat straw or oat spelt xylan produced the highest titres of xylanse and beta-xylosidase. None of the soluble carbon sources investigated produced the high titres of xylanase or beta-xylosidase induced by either oat straw for xylanse and beta-xylosidase was 2% and the optimum spore inoculum was between 10(6) and 10(7) spores/mL(-1) final concentration. The level of xylanse activity obtained in the culture filtrates of the mutant was a remarkable 820 U mL(-1) when the reducing sugar released was measured by the dinitrosalicylic acid method. This enzyme titre would appear to be the highest reported so far. The xylanases system contained the correct balance of enzymes to effect extensive hydrolysis of oat spelt xylan. The protease titre was very low.     

Corynebacterium Pseudotuberculosis Infection in Goats I.

The bacterial agglutination test (BAT) and the hemolysis inhibition test (HIT) were employed and evaluated. The threshold value between positive and negative antibody titres in BAT and HIT was determined on the basis of titration results for sera from 25 adult goats infected with Corynebacterium pseudotuber-culosis (positive), and sera from 25 adult goats from a herd in which caseous lymphadenitis did not occur (negative). Antibody titres were expressed as logio to the reciprocal value of the highest positive serum dilution in both tests. Positive titre (T) in BAT was stipulated as T ≥ 2.1 and in HIT as T > 0.6. The sensitivity and specificity was 0.96 in both tests in the material used. Twenty-three of the 25 goats infected with G. pseudotuberculosis were positive in both BAT and HIT. The reproducibility in both tests was described by the use of correlation coefficient and coefficient of variation. The values were estimated using duplicate determination of titres of 155 serum samples. Correlation coefficients were 0.87 for BAT and 0.95 for HIT. Coef-ficients of variation were 26.4 % for BAT and 14.1 % for HIT. The coefficient of variation was related to titres. It was highest for BAT at low titres. It was concluded that both tests can be used for seroepidemio-logical investigations of C. pseudotuberculosis infection in goats.     

Persistence of antibodies of the IgG class against cytomegaloviruses in blood donors

High titre plasmas gained from blood donors are the initial material used for producing human immunoglobulin containing cytomegalovirus antibodies (CMV-HIG). For this purpose the sera gained from 467 permanent donors at the District Institute for Blood and Transfusion Service in Berlin were investigated on their CMV antibody content of IgG class by means of an indirect immunofluorescence test (IFT). The infection rate of blood donors amounted to 62% (291/467). CMV-IgG titre greater than or equal to 1:40 was determined in 88 sera (18.8%) and greater than or equal to 1:160 in 14 sera (3%). Two CMV-HIG laboratory samples (charges 3113 and 3117) were produced from these plasmas. Particularly immunoglobulin fraction of charge 3117 (No. 3117 N II) revealed excellent antibody titres (IFT 1:640 [CMV-IgG] or 1:40 [CMV-IgM] respectively, neutralisation test 1:32). Checks made with the donors' CMV-IgG titres after repeated application of plasmapheresis resulted in maximal titres changes of two dilution stages in a period of 15 months. Thus, in producing CMV-HIG a sure, tested pool of donors can be resorted to.     

Evaluating the mean frequency of responding cells in limiting dilution assays

Summary The validity of limiting dilution assays can be compromised or negated by the use of statistical methodology which does not consider all issues surrounding the biological process. This study critically evaluates statistical methods for estimating the mean frequency of responding cells in multiple sample limiting dilution assays. We show that methods that pool limiting dilution assay data, or samples, are unable to estimate the variance appropriately. In addition, we use Monte Carlo simulations to evaluate an unweighted mean of the maximum likelihood estimator, an unweighted mean based on the jackknife estimator, and a log transform of the maximum likelihood estimator. For small culture replicate size, the log transform outperforms both unweighted mean procedures. For moderate culture replicate size, the unweighted mean based on the jackknife produces the most acceptable results. This study also addresses the important issue of experimental design in multiple sample limiting dilution assays. In particular, we demonstrate that optimization of multiple sample limiting dilution assays is achieved by increasing the number of biological samples at the expense of repeat cultures.     

Chlamydophila pneumoniae in horses: a seroepidemiological survey in Italy

We tested 731 sera from apparently healthy light horses against Chlamydophila pneumoniae, by a microimmuno-fluorescence (MIF) test. To verify cross-reactions with other species of chlamvdiae, all sera with an antibody titre > or = 32 to C. pneumoniae were tested against both C. psittaci and C. abortus. Antibodies to C. pneumoniae were detected in 194 out of 731 (26.5%) samples tested, with antibody titres ranging from 32 to 1024. No antibody titre > or = 32 was detected in sera to C. abortus. Only few sera with a high antibody titre to C. pneumoniae reacted weakly with C. psittaci at the dilution of 1:32.     

流行性出血热病毒沙鼠肾细胞培养灭活疫苗免疫家兔的抗体反应及保护试验

利用自流行性出血热(EHF)病人血液中分离的EHF病毒浙10株,感染长爪沙鼠肾单层细胞,经培养后,按流行性乙型脑炎的生物制品法规要求研制成灭活疫苗。取1ml经肌肉免疫家兔,6天后即可在血液中检出荧光抗体,至第14～21天为最高峰,荧光抗体效价可达320～2560。经活EHF病毒攻击,有明显的保护作用,保护率达90％以上。疫苗经1:10或1:30稀释后免疫家兔,荧光抗体阳转率仍达100％,但抗体滴度明显降低。肌肉接种优于皮下接种。本研究证明,甲醛灭活EHF病毒可破坏血凝素活性,从而影响疫苗血凝抑制抗体的产生,可能也会影响中和病毒的能力。     

Early kinetics of antibody response to inactivated influenza vaccine

The aim was to examine the rapidity of haemagglutination inhibiting (HI) antibody response induced by immunization with a current inactivated trivalent influenza vaccine. Five to six sequential serum samples collected in autumn 1992 from each of 68 vaccinees in three age groups were studied for HI antibodies to ten influenza virus strains representing vaccine and epidemic viruses. Geometric mean titres, response rates and protection rates are presented. Response rates of > 70% were overall, but not until two weeks after the vaccination. Significant two- and four-day post-vaccination antibody responses were detected only occasionally. In previously vaccinated persons, average antibody titres to some of the viruses decreased during the first days after the vaccination. In the subsequent samples, the titres remained lower than in persons who were not vaccinated against influenza in preceding years. Protection against influenza infection may be frequently developed not until two weeks after vaccination. This has relevance to prophylactic administration of amantadine and rimantadine when an influenza A outbreak is imminent and the vaccination is late.     

Maximum likelihood estimation of growth yields

This work is concerned with statistical methods to estimate yield and maintenance parameters associated with microbial growth. For a given dilution rate, an experimenter typically measures substrate concentration, oxygen utilization rate, the rate of carbon dioxide evolution, and biomass concentration. These correlated response variables each contain information about the maintenance and yield parameters of interest. A maximum likelihood estimator which combines this correlated information for the yield and maintenance parameters is proposed, evaluated, and tested on literature data. Both point and interval estimators are considered.     

Production and biological activity of murine monoclonal antibodies against GnRH

Immunization against GnRH represents a nonsurgical means of castrating domestic species. However, clear target antibody titres for bioactivity have not been established. The aims of this study were to produce characterized anti-GnRH monoclonal antibodies and to determine a threshold titre. Three murine monoclonals were developed which produced IgG2a class immunoglobulins and bound 50% I(125)-GnRH at a 10(6) to 10(7) dilution. The antibodies were specific to GnRH, showed a strong affinity (Ka values from 1.99 to 2.60 x 10(10) litres/mole), and were directed towards the amino terminus. In female mice all 3 antibody clones interrupted ovarian cyclicity, causing an extension in diestrus followed by prolonged estrus/metestrus (12 to 30 d). Throughout this period circulating titres were greater than 15% I(125)-GnRH binding at a 5 x 10(4) dilution. In male mice, immunization with 0.2 ml of ascites significantly reduced testes (P < 0.05), epididymides (P < 0.001) and seminal vesicle (P < 0.01) weights. A 0.1 ml dose (61.4 +/- 18.6% binding at a 10(6) dilution) was ineffective. A serial dilution study indicated that a titre of 50% binding at 2 x 10(6) dilution (antigen binding capacity of 268 +/- 35 ng/ml) was required to completely block GnRH activity. This is a higher tire than threshold levels determined previously. Identification of factors determining the titre required for bioactivity is needed.     

Synthesis of natural allohemagglutinins of the ABO system in healthy children aged 3 months to 3 years

The work was carried out to establish the titre and score of haemagglutination of natural anti-A and anti-B antibodies in healthy children during the first three years of life. The material studied included 900 healthy children aged between 3 months and 3 years and 100 adults serving as controls. The method of test tube haemagglutination was used for determining the titre and score of alloagglutinins in relation to standard erythrocytes always obtained from the same donors. In addition, in 72 children and 10 adults the levels of IgG, IgA and IgM were determined quantitatively. A statistical analysis of the results showed that the levels of anti-A and anti-B alloagglutinins were relatively high at the age of 3 months / about 25% of the adult levels / and increased very rapidly in the first years of life reaching about 90% of the adult level at the end of the 3rd year of life. Besides that, it was demonstrated that it is useful in clinical practice to use the titre and score of natural alloagglutinins as indicators of humoral immunity, especially in children in the first years of life. Acceleration was demonstrated in the intensity of haemagglutination of natural antibodies in the last 40 years since their titre in the contemporary infantile population / up to the age of 1 year / is about 50% higher than that found in 1929. These findings suggest that increased immune reactivity of children observed presently may be due to prophylactic vaccinations.     

Comparison of serological assays for detecting antibodies in ducks exposed to H5 subtype avian influenza virus

ABSTRACT: BACKGROUND: Chicken red blood cells (RBCs) are commonly used in hemagglutination inhibition (HI) tests to measure hemagglutinating antibodies against influenza viruses. The use of horse RBCs in the HI test can reportedly increase its sensitivity when testing human sera for avian influenza antibodies. This study aims to compare the proportion of positives detected and the agreement between two HI tests using either chicken or horse red blood cells for antibody detection in sera of ducks experimentally infected or naturally exposed to Indonesian H5 subtype avian influenza virus. In addition, comparison with a virus neutralisation (VN) test was conducted with the experimental sera. RESULTS: In the experimental study, the proportion of HI antibody-positive ducks increased slightly, from 0.57 when using chicken RBCs to 0.60 when using horse RBCs. The HI tests indicated almost perfect agreement (kappa = 0.86) when results were dichotomised (titre [greater than or equal to] 4 log2), and substantial agreement (weighted kappa = 0.80) for log titres. Overall agreements between the two HI tests were greater than between either of the HI tests and the VN test. The use of horse RBCs also identified a higher proportion of antibody positives in field duck sera (0.08, compared to chicken RBCs 0.02), with also almost perfect agreements for dichotomized results (Prevalence and bias adjusted Kappa (PABAK) = 0.88) and for log titres (weighted PABAK = 0.93), respectively. Factors that might explain observed differences in the proportion of antibody-positive ducks and in the agreements between HI tests are discussed. CONCLUSION: In conclusion, we identified a good agreement between HI tests. However, when horse RBCs were used, a higher proportion of sera was positive (titre [greater than or equal to] 4 log2) than using chicken RBCs, especially during the early response against H5N1 virus. The HRBC-HI might be more responsive in identifying early H5N1 HPAI serological response and could be a recommended assay for avian influenza sero-surveillance in both wild and domestic birds.     

Indirect haemagglutination reaction (LH)-the method of choice for the detection of anamnestic antibodies to varicella - zoster (VZ) virus.

The method of indirect haemagglutination is a sensitive quantitative serological reaction for the detection of anamnestic titres of VZ antibodies. The curve obtained by the examination of a representative population sample has shown that the occurrence of VZ antibodies increases with the age so that 60% of children of under-school age and 90% of 15-year-old children have antibodies to varicella-zoster virus. In adult age groups, seropositivity ranges between 90--100%. The incidence and level of antibodies are identical in both sexes and do not decrease in old age groups. The mean titre of indirect haemagglutinating antibodies was 1 : 128.     

Estimation in the cox proportional hazards model with left-truncated and interval-censored data

We show that the nonparametric maximum likelihood estimate (NPMLE) of the regression coefficient from the joint likelihood (of the regression coefficient and the baseline survival) works well for the Cox proportional hazards model with left-truncated and interval-censored data, but the NPMLE may underestimate the baseline survival. Two alternatives are also considered: first, the marginal likelihood approach by extending Satten (1996, Biometrika 83, 355-370) to truncated data, where the baseline distribution is eliminated as a nuisance parameter; and second, the monotone maximum likelihood estimate that maximizes the joint likelihood by assuming that the baseline distribution has a nondecreasing hazard function, which was originally proposed to overcome the underestimation of the survival from the NPMLE for left-truncated data without covariates (Tsai, 1988, Biometrika 75, 319-324). The bootstrap is proposed to draw inference. Simulations were conducted to assess their performance. The methods are applied to the Massachusetts Health Care Panel Study data set to compare the probabilities of losing functional independence for male and female seniors.     

Prevalence of antibodies to 2009 pandemic influenza A (H1N1) virus in German adult population in pre- and post-pandemic period

Background

In order to detect levels of pre-existing cross-reactive antibodies in different age groups and to measure age-specific infection rates of the influenza A (H1N1) 2009 pandemic in Germany, we conducted a seroprevalence study based on samples from an ongoing nationwide representative health survey.

Methodology/Principal Findings

We analysed 845 pre-pandemic samples collected between 25 Nov 2008 and 28 Apr 2009 and 757 post-pandemic samples collected between 12 Jan 2010 and 24 Apr 2010. Reactive antibodies against 2009 pandemic influenza A (H1N1) virus (pH1N1) were detected using a haemagglutination inhibition test (antigen A/California/7/2009). Proportions of samples with antibodies at titre ≥40 and geometric mean of the titres (GMT) were calculated and compared among 6 age groups (18–29, 30–39, 40–49, 50–59, 60–69, ≥70 years). The highest proportions of cross-reactive antibodies at titre ≥40 before the pandemic were observed among 18–29 year olds, 12.5% (95% CI 7.3–19.5%). The highest increase in seroprevalence between pre- and post-pandemic was also observed among 18–29 year olds, 29.9% (95% CI 16.7–43.2%). Effects of sampling period (pre- and post-pandemic), age, sex, and prior influenza immunization on titre were investigated with Tobit regression analysis using three birth cohorts (after 1976, between 1957 and 1976, and before 1957). The GMT increased between the pre- and post-pandemic period by a factor of 10.2 (95% CI 5.0–20.7) in the birth cohort born after 1976, 6.3 (95% CI 3.3–11.9) in those born between 1957 and 1976 and 2.4 (95% CI 1.3–4.3) in those born before 1957.

Conclusions/Significance

We demonstrate that infection rates differed among age groups and that the measured pre-pandemic level of cross-reactive antibodies towards pH1N1 did not add information in relation to protection and prediction of the most affected age groups among adults in the pandemic.     

Detection and titration of anti-tetanus antibodies by passive hemagglutination]

Human red cells O Rh + are coated with purified tetanus toxoid, by means of chromic chloride as coupling agent. With this reagent and by passive haemagglutination technique, antitetanus antibodies can be automatically screened and titrated in blood donnors plasmas, on Groupamatic equipments. Comparative results of three techniques: counter immunoelectrophoresis; manual passive haemagglutination; automatic passive haemagglutination are given, 4, 92 percent among the 1.219 tested blood donnors plasmas have an antibody greater than or equal to 5 U.I., 16, 24 percent have a titre between 1 and 5 U.I.     

Pandemic Influenza A(H1N1)pdm09 Seroprevalence in Sweden before and after the Pandemic and the Vaccination Campaign in 2009

The immunity to pandemic influenza A(H1N1)pdm09 in Sweden before and after the outbreaks in 2009 and 2010 was investigated in a seroepidemiological study. Serum samples were collected at four time points: during 2007 (n = 1968), in October 2009 (n = 2218), in May 2010 (n = 2638) and in May 2011 (n = 2513) and were tested for hemagglutination inhibition (HI) antibodies. In 2007, 4.9% of the population had pre-existing HI titres ≥40, with the highest prevalence (20.0%) in 15–24 year-olds, followed by ≥80 year-olds (9.3%). The overall prevalence of HI titres ≥40 had not changed significantly in October 2009. In May 2010 the prevalence had increased to 48.6% with the highest percentages in 5–14 year-olds (76.2%) andlowest in 75–79 year-olds (18.3%). One year later the prevalence of HI titres ≥40 had increased further to 52.2%. Children 5–14 years had the highest incidence of infection and vaccine uptake as well as the highest post-pandemic protective antibody levels. In contrast, the elderly had high vaccine uptake and low attack rate but low levels of protective antibodies, underlining that factors other than HI antibodies are involved in protection against influenza A(H1N1)pdm09. However, for all age-groups the seroprevalence was stable or increasing between 2010 and 2011, indicating that both vaccine- and infection-induced antibodies were long-lived.