Antibody Titers In Serum Samples Research Articles

Correlating IgG Levels with Neutralising Antibody Levels to Indicate Clinical Protection in Healthcare Workers at Risk during a Measles Outbreak.

The rapid transmission of measles poses a great challenge for measles elimination. Thus, rapid testing is required to screen the health status in the population during measles outbreaks. A pseudotype-based virus neutralisation assay was used to measure neutralising antibody titres in serum samples collected from healthcare workers in Sheffield during the measles outbreak in 2016. Vesicular stomatitis virus (VSV) pseudotypes bearing the haemagglutinin and fusion glycoproteins of measles virus (MeV) and carrying a luciferase marker gene were prepared; the neutralising antibody titre was defined as the dilution resulting in 90% reduction in luciferase activity. Spearman’s correlation coefficients between IgG titres and neutralising antibody levels ranged from 0.40 to 0.55 (p < 0.05) or from 0.71 to 0.79 (p < 0.0001) when the IgG titres were obtained using different testing kits. In addition, the currently used vaccine was observed to cross-neutralise most circulating MeV genotypes. However, the percentage of individuals being “well-protected” was lower than 95%, the target rate of vaccination coverage to eliminate measles. These results demonstrate that the level of clinical protection against measles in individuals could be inferred by IgG titre, as long as a precise correlation has been established between IgG testing and neutralisation assay; moreover, maintaining a high vaccination coverage rate is still necessary for measles elimination.

The use of dried blood spot cards to assess serologic responses of individuals vaccinated against measles, hepatitis A, tetanus, influenza and varicella zoster.

Traditional blood sampling by venipuncture is cumbersome and relatively expensive. Dried blood spot (DBS) sampling is desirable because of its ease of sample collection, transportation and storage. It has been used in clinical diagnosis but not been thoroughly studied for the potential use to assess the immune status of individuals following natural infection or preventive vaccination. The goal of this study was to compare DBS to traditional blood samplings in detection of antibodies in individuals vaccinated against measles, hepatitis A, tetanus, influenza and varicella zoster. Enzyme linked immunosorbent assay (ELISA) was used to test DBS eluates and serum samples for antibodies against measles, varicella, tetanus and hepatitis A. Sensitivities, specificities, and correlation coefficients were evaluated to compare optical density (OD) values of paired serum and DBS samples. The long-term stability of DBS samples at different temperatures was assessed using simulated immune measles blood. DBS OD was highly correlated with serum OD for antibodies to measles (r = 0.93), varicella (r = 0.82), and tetanus (r = 0.91). Sensitivities of DBS OD ranged from 86–99% and specificities ranged from 96–100% using cut-offs established by each assay. By contrast, the hepatitis A data showed a low sensitivity (31%) and weak correlation (r = 0.14) between DBS and serum samples. Antibody titers in serum samples for anti-influenza A (H1N1 and H3N1) failed to correlate in DBS eluates in HAI and MN assays. DBS samples were stable for 4 weeks when stored at room temperature and for 6 months at 4°C. DBS sampling was sensitive, specific, and highly correlated with traditional venipuncture sampling in detection of antibodies against measles, tetanus and varicella zoster, but not hepatitis A and influenza. Thus, the success of using DBS sampling to assess the antibody levels in immunized individuals may be dependent on the pathogens and the development of the assay used.

Evaluation of the effect of maternally derived antibody on response to MMR vaccine in Thai infants

BackgroundAlthough the number of measles cases declined globally in response to anti-measles immunisation campaigns, measles has re-emerged. A review of current vaccination policies is required to improve measles elimination strategies. MethodsA pseudotype-based virus neutralisation assay (PVNA) was used to measure neutralising antibody titres in serum samples collected from Thai infants at six timepoints before and after two-doses of MMR (1&2) vaccination (ClinicalTrials.gov no. NCT02408926). Vesicular stomatitis virus (VSV) luciferase pseudotypes bearing the haemaglutinin (H) and fusion (F) glycoproteins of measles virus (MeV) were prepared. Serial dilutions of serum samples were incubated with VSV (MeV) pseudotypes and plated onto HEK293-human SLAM1 cells; the neutralising antibody titre was defined as the dilution resulting in 90% reduction in luciferase activity. ResultsNeutralising antibody titres in infants born with high levels of maternal immunity (H group) persisted at the time of the first MMR vaccination, and those infants did not respond effectively by developing protective titres. In contrast, infants with lower maternal immunity (L group) developed protective titres of antibody following vaccination. Responses to the second MMR vaccination were significantly higher (P = 0.0171, Wilcoxon signed-rank test) in the H group. The observed correlation between anti-MeV IgG level and neutralising antibody titre in Thai infants indicates the possibility of using rapid IgG testing as a surrogate measure for neutralising activity to define clinical protection levels within populations. ConclusionThese results demonstrate that varying the timing of the first MMR immunisation according to the level of acquired maternal immunity could increase vaccination immunogenicity and hence accelerate measles eradication.

Detection of Serum Cross-Reactive Antibodies and Memory Response to SARS-CoV-2 in Prepandemic and Post-COVID-19 Convalescent Samples.

BackgroundA notable feature of coronavirus disease 2019 (COVID-19) is that children are less susceptible to severe disease. Children are known to experience more infections with endemic human coronaviruses (HCoVs) compared to adults. Little is known whether HCoV infections lead to cross-reactive anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies.MethodsWe investigated the presence of cross-reactive anti–SARS-CoV-2 IgG antibodies to spike 1 (S1), S1-receptor-binding domain (S1-RBD), and nucleocapsid protein (NP) by enzyme-linked immunosorbent assays, and neutralizing activity by a SARS-CoV-2 pseudotyped virus neutralization assay, in prepandemic sera collected from children (n = 50) and adults (n = 45), and compared with serum samples from convalescent COVID-19 patients (n = 16).ResultsA significant proportion of children (up to 40%) had detectable cross-reactive antibodies to SARS-CoV-2 S1, S1-RBD, and NP antigens, and the anti-S1 and anti–S1-RBD antibody levels correlated with anti–HCoV-HKU1 and anti–HCoV-OC43 S1 antibody titers in prepandemic samples (P < .001). There were marked increases of anti–HCoV-HKU1 and - OC43 S1 (but not anti-NL63 and -229E S1-RBD) antibody titers in serum samples from convalescent COVID-19 patients (P < .001), indicating an activation of cross-reactive immunological memory to β-coronavirus spike.ConclusionsWe demonstrated cross-reactive anti–SARS-CoV-2 antibodies in prepandemic serum samples from children and young adults. Promoting this cross-reactive immunity and memory response derived from common HCoV may be an effective strategy against SARS-COV-2 and future novel coronaviruses.

A high throughput reporter virus particle microneutralization assay for quantitation of Zika virus neutralizing antibodies in multiple species.

Zika virus is a Flavivirus, transmitted via Aedes mosquitos, that causes a range of symptoms including Zika congenital syndrome. Zika has posed a challenging situation for health, public and economic sectors of affected countries. To quantitate Zika virus neutralizing antibody titers in serum samples, we developed a high throughput plate based Zika virus reporter virus particle (RVP) assay that uses an infective, non-replicating particle encoding Zika virus surface proteins and capsid (CprME) and a reporter gene (Renilla luciferase). This is the first characterization of a Zika virus RVP assay in 384-well format using a Dengue replicon Renilla reporter construct. Serially diluted test sera were incubated with RVPs, followed by incubation with Vero cells. RVPs that have not been neutralized by antibodies in the test sera entered the cells and expressed Renilla luciferase. Quantitative measurements of neutralizing activity were determined using a plate-based assay and commercially available substrate. The principle of limiting the infection to a single round increases the precision of the assay measurements. RVP log10EC50 titers correlated closely with titers determined using a plaque reduction neutralization test (PRNT) (R2>95%). The plate-based Zika virus RVP assay also demonstrated high levels of precision, reproducibility and throughput. The assay employs identical reagents for human, rhesus macaque and mouse serum matrices. Spiking studies indicated that the assay performs equally well in different species, producing comparable titers irrespective of the serum species. The assay is conducted in 384-well plates and can be automated to simultaneously achieve high throughput and high reproducibility.

Temperature and Time Dependent Variations in Anti-Toxoplasma gondii Antibody Titrations in Serum Samples

This study aimed to determine the temperature- and time-dependent variations in the anti-Toxoplasma gondii (T. gondii) antibody titres in serum samples collected from T. gondii-infected patients. During May 2017-February 2018, five serum samples (stored at different time periods and conditions) from pregnant or non-pregnant women aged ≥18 years who were infected with T. gondii and had applied to our Parasitology Department of Hafsa Sultan Hospital, Manisa Celal Bayar University, were investigated for the anti-T. gondii IgG antibody levels by enzyme linked fluorescent assay (ELFA). The serum samples of five female volunteers who were infected with T. gondii that were stored at room temperature (20/25 °C), in a cargo package (+4/+8 °C), in a refrigerator (+4 °C), in a deep freezer (-16/-20 °C) and in an incubator (+37 °C) were tested at 0, 24, 48 and 72 hours after infection with the ELFA test. No statistically significant difference was observed in the anti-T. gondii IgG antibody titres (p>0.05). The results obtained from the patients infected with T. gondii at different times and conditions of up to 72 hours were not significantly affected clinically. Hence, more comprehensive data can be obtained by increasing the number of patients and storing the serum samples for more than 72 hours.

151. The Use of Dried Blood Spot Cards to Assess Serologic Responses of Individuals Vaccinated Against Measles, Hepatitis A, Tetanus, Influenza and Varicella Zoster

BackgroundTraditional blood sampling by venipuncture is cumbersome and expensive. Dried Blood Spot (DBS) sampling is desirable because of its ease of sample collection, transportation and storage. It has been used in clinical diagnosis but not been thoroughly studied for the potential use to assess the immune status of individuals following natural infection or preventive vaccination. The goal of this study is to compare DBS to traditional blood samplings in detection of antibodies in individuals vaccinated against measles, hepatitis A, tetanus, influenza and varicella zoster.MethodsBlood from 220 vaccinated individuals were collected by venipuncture into serum separation tubes and by fingerstick onto Whatman 903® protein saver cards. ELISA was used to test DBS eluates and serum samples for antibodies against measles, varicella, tetanus and hepatitis A. Sensitivities, specificities, and correlation coefficients were evaluated to compare optical density (OD) values of paired serum and DBS samples. Hemagglutinin inhibition (HAI) and microneutralization assay (MNA) were used to determine anti-influenza antibody in serum and DBS samples. The long term stability of DBS samples at different temperatures was assessed using simulated immune measles blood.ResultsDBS OD was highly correlated with serum OD for antibodies to measles (r = 0.93), varicella (r = 0.82), and tetanus (r = 0.91) (Fig.1). Sensitivities of DBS OD ranged from 86–99% and specificities ranged from 96–100% using cut-offs established by each assay. By contrast, the hepatitis A data showed a low sensitivity (31%) and a weak correlation (r = 0.14) between DBS and serum samples. HAI and MNA assays showed a broad range of anti-influenza A (H1N1 and H3N1) antibody titers in serum samples but failed to detect the antibodies in DBS eluates. The stability data indicated that DBS samples were stable for 4 weeks when stored at room temperature and for 6 months at 4°C (Fig. 2).Figure 1. Correlation of antibody levels detected by DBS sampling to the antibody levels detected by serum sampling for measles, hepatitis A, tetanus and varicella zoster. Figure 2. Stability of the blood samples on DBS cards stored under various temperatures for 25 weeks as measured by the titers of anti-measles antibody (IgG) at various time points. ConclusionDBS sampling was sensitive, specific, and highly correlated with traditional venipuncture sampling in detection of antibodies against measles, tetanus and varicella zoster, but not hepatitis A and influenza. Thus, the success of using DBS sampling to assess the antibody levels in immunized individuals may be dependent on the pathogens and the type of assay used.Disclosures All Authors: No reported disclosures

A time-resolved fluoroimmunoassay for assessing rabies antibody titers in the sera of vaccinated human subjects

Several studies have investigated the use of simple in vitro tests for the assessment of rabies antibody titers in serum samples from vaccinated human subjects, which would allow the effectiveness of rabies vaccination to be conveniently evaluated. To this end, a novel time-resolved fluoroimmunoassay (TRFIA) for the assessment of rabies antibody titers was established in this study for evaluating the effectiveness of protection against rabies. The TRFIA had a satisfactory limit of detection value (0.035 IU/mL) under optimal conditions. Additionally, the application of the TRFIA was demonstrated in 68 serum samples with satisfactory results. The coefficient variations (CVs) were all <10%, and the recoveries were in the range of 90–110%. The correlation coefficient of titer values obtained using the present TRFIA and the rapid fluorescent focus inhibition test (RFFIT) was 0.733, with a coincidence rate regarding the evaluation results (protected or not protected by vaccination) of 100%. The preliminary results confirmed that the TRFIA had a higher performance than an enzyme-linked immunosorbent assay (ELISA), and could potentially replace the ELISA. Based on these results, the novel TRFIA appears to be a convenient tool for the evaluation of rabies vaccination results based on serum samples from vaccinated human subjects.

Human immune responses elicited by an intranasal inactivated H5 influenza vaccine.

Intranasally administered influenza vaccines could be more effective than injected vaccines, because intranasal vaccination can induce virus‐specific immunoglobulin A (IgA) antibodies in the upper respiratory tract, which is the initial site of infection. In this study, immune responses elicited by an intranasal inactivated vaccine of influenza A(H5N1) virus were evaluated in healthy individuals naive for influenza A(H5N1) virus. Three doses of intranasal inactivated whole‐virion H5 influenza vaccine induced strong neutralizing nasal IgA and serum IgG antibodies. In addition, a mucoadhesive excipient, carboxy vinyl polymer, had a notable impact on the induction of nasal IgA antibody responses but not on serum IgG antibody responses. The nasal hemagglutinin (HA)‐specific IgA antibody responses clearly correlated with mucosal neutralizing antibody responses, indicating that measurement of nasal HA‐specific IgA titers could be used as a surrogate for the mucosal antibody response. Furthermore, increased numbers of plasma cells and vaccine antigen‐specific Th cells in the peripheral blood were observed after vaccination, suggesting that peripheral blood biomarkers may also be used to evaluate the intranasal vaccine‐induced immune response. However, peripheral blood immune cell responses correlated with neutralizing antibody titers in serum samples but not in nasal wash samples. Thus, analysis of the peripheral blood immune response could be a surrogate for the systemic immune response to intranasal vaccination but not for the mucosal immune response. The current study suggests the clinical potential of intranasal inactivated vaccines against influenza A(H5N1) viruses and highlights the need to develop novel means to evaluate intranasal vaccine‐induced mucosal immune responses.

ANTIBODY RESPONSE TO SEVERAL MALARIA ANTIGENS IS ASSOCIATED WITH PROTECTION FROM SEVERE MALARIA IN UGANDAN CHILDREN

BackgroundMalaria is one of the leading causes of morbidity. Both natural infection and irradiated sporozoites have yielded partial and sterile immunity and contain multiple antigens so a malaria vaccine with...

Benefits and Limits of Egg Yolkvs. Serum Samples for Avian Influenza Virus Serosurveillance

Serologic tests are a valuable tool for retrospective surveillance of avian influenza viruses (AIV) and monitoring of postvaccination host immune response. Yet collection of serum samples, particularly in adult breeder chickens, is laborious, intrusive to birds, and may pose a serious risk to the biosecurity of a flock. In this study we compared the level of AIV-specific antibody titers in eggs and serum samples obtained from broiler breeder chickens vaccinated at 6, 12, and 18 wk of age with H5N2-inactivated vaccine. Nucleocapsid protein-specific ELISA and hemagglutination inhibition test (HI) against homologous as well as heterologous antigens were used. The eggs and sera were collected at 22, 30, 45, and 50 wk of age (i.e., 4, 12, 27, and 32 wk after the third and final immunization, respectively). Using ELISA, the number of positive egg yolk samples decreased over time after vaccination, from 97% to 47%, while the seropositivity rate of serum samples was 97%-100% during the whole investigation period. No antibody titers were detected in egg white. By HI, antibody titers in serum samples were higher than in egg yolk samples. Compared to the homologous H5N2 antigen, significantly lower HI titers were obtained by using a heterologous H5N1 virus of clade 2.2.1.2. In addition, no HI titers were detected in egg yolk and/or serum samples tested against the antigen of an Egyptian H5N1 antigenic drift variant of clade 2.2.1.1. This study indicates that egg yolk may be used to monitor the postvaccination immune status of broiler breeder chickens and retrospective serosurveillance-by HI when a matching antigen is available as well as by ELISA-particularly for up to 12 wk postvaccination.

Seroprevalence of Antibodies against Seal Influenza A(H10N7) Virus in Harbor Seals and Gray Seals from the Netherlands.

In the spring and summer 2014, an outbreak of seal influenza A(H10N7) virus infection occurred among harbor seals (Phoca vitulina) off the coasts of Sweden and Denmark. This virus subsequently spread to harbor seals off the coasts of Germany and the Netherlands. While thousands of seals were reported dead in Sweden, Denmark and Germany, only a limited number of seals were found dead in the Netherlands. To determine the extent of exposure of seals in the Netherlands to influenza A/H10N7 virus, we measured specific antibody titers in serum samples from live-captured seals and seals admitted for rehabilitation in the Netherlands by use of a hemagglutination inhibition assay and an ELISA. In harbor seals in 2015, antibodies against seal influenza A(H10N7) virus were detected in 41% (32 out of 78) pups, 10% (5 out of 52) weaners, and 58% (7 out of 12) subadults or adults. In gray seals (Halichoerus grypus) in 2015, specific antibodies were not found in the pups (n = 26), but in 26% (5 out of 19) of the older animals. These findings indicate that, despite apparent low mortality, infection with seal influenza A(H10N7) virus was geographically widespread and also occurred in grey seals.

Immunization of Mice with Anthrax Protective Antigen Limits Cardiotoxicity but Not Hepatotoxicity Following Lethal Toxin Challenge.

Protective immunity against anthrax is inferred from measurement of vaccine antigen-specific neutralizing antibody titers in serum samples. In animal models, in vivo challenges with toxin and/or spores can also be performed. However, neither of these approaches considers toxin-induced damage to specific organ systems. It is therefore important to determine to what extent anthrax vaccines and existing or candidate adjuvants can provide organ-specific protection against intoxication. We therefore compared the ability of Alum, CpG DNA and the CD1d ligand α-galactosylceramide (αGC) to enhance protective antigen-specific antibody titers, to protect mice against challenge with lethal toxin, and to block cardiotoxicity and hepatotoxicity. By measurement of serum cardiac Troponin I (cTnI), and hepatic alanine aminotransferase (ALT), and aspartate aminotransferase (AST), it was apparent that neither vaccine modality prevented hepatic intoxication, despite high Ab titers and ultimate survival of the subject. In contrast, cardiotoxicity was greatly diminished by prior immunization. This shows that a vaccine that confers survival following toxin exposure may still have an associated morbidity. We propose that organ-specific intoxication should be monitored routinely during research into new vaccine modalities.

The relationship between Helicobacter pylori seropositivity and COPD

RationaleChronic systemic infections such as those with Helicobacter pylori (H. pylori) may contribute to the evolution and progression of chronic obstructive pulmonary disease (COPD). Using data from the Lung Health...

Peptide mimotopes of malondialdehyde-epitopes for clinical applications in cardiovascular disease

IntroductionAutoantibodies specific for malondialdehyde modified LDL (MDA-LDL) represent potential biomarkers to predict cardiovascular risk. However, the generation of MDA-LDL results in the formation of many different epitopes with high variability. Because it is not known, which MDA epitope is biological important, the aim of this study was to identify and characterize peptide mimotopes of MDA-LDL that could be used as antigens to improve the reproducible detection of MDA-specific autoantibodies. Methods and ResultsPeptide phage display libraries were screened for phages binding to the MDA-LDL specific natural IgM antibody LR04. After biopanning two consensus sequences (P1 & P2) of binding phages were synthesized. P1 and P2 were specifically bound by LR04, as the binding of LR04 to coated peptides was fully competed by MDA-LDL but not native LDL. P1 and P2 were also bound by other MDA specific murine (EN1) and human (IK17) antibodies. Furthermore, the binding of LR04 to late apoptotic cells was completely inhibited by both peptides, identifying them as mimotopes of naturally occurring epitopes on dying cells. Immunization of C57BL/6 mice with P2 conjugated to BSA, but not BSA alone, resulted in the robust induction of IgG1 and IgM antibodies against MDA-LDL. Moreover, serum IgG of immunized mice specifically stained epitopes in atherosclerotic lesion of rabbits and humans. Finally, we measured anti-mimotope antibody titers in serum samples previously collected from healthy subjects (n=17) and from patients (n=140) with stable angina pectoris undergoing percutaneous coronary intervention (Tsimikas, 2004). In patients a significant positive correlation was observed between anti-MDA-LDL and anti-mimotope IgM (P1, r=0.8; P2, r=0.6; p<0.0001) and IgG (P1, r=0.4; P2, r=0.3; p<0.0001) antibodies. A similar correlation was also found in sera of healthy subjects with IgM (P1, r=0.6; P2, r=0.4; p<0.0004) and IgG (P1, r=0.7; P2, r=0.5; p<0.0001) antibodies. ConclusionsThus, we have identified specific mimotopes of MDA-LDL that serve as highly reproducible antigens to assess autoantibody titers in patients with cardiovascular disease. Future studies will reveal their usefulness for therapeutic vaccination approaches against atherosclerosis.

Toxoplasma gondii 70 kDa Heat Shock Protein: Systemic Detection Is Associated with the Death of the Parasites by the Immune Response and Its Increased Expression in the Brain Is Associated with Parasite Replication

The heat shock protein of Toxoplasma gondii (TgHSP70) is a parasite virulence factor that is expressed during T. gondii stage conversion. To verify the effect of dexamethasone (DXM)-induced infection reactivation in the TgHSP70-specific humoral immune response and the presence of the protein in the mouse brain, we produced recombinant TgHSP70 and anti-TgHSP70 IgY antibodies to detect the protein, the specific antibody and levels of immune complexes (ICs) systemically, as well as the protein in the brain of resistant (BALB/c) and susceptible (C57BL/6) mice. It was observed higher TgHSP70-specific antibody titers in serum samples of BALB/c compared with C57BL/6 mice. However, the susceptible mice presented the highest levels of TgHSP70 systemically and no detection of specific ICs. The DXM treatment induced increased parasitism and lower inflammatory changes in the brain of C57BL/6, but did not interfere with the cerebral parasitism in BALB/c mice. Additionally, DXM treatment decreased the serological TgHSP70 concentration in both mouse lineages. C57BL/6 mice presented high expression of TgHSP70 in the brain with the progression of infection and under DXM treatment. Taken together, these data indicate that the TgHSP70 release into the bloodstream depends on the death of the parasites mediated by the host immune response, whereas the increased TgHSP70 expression in the brain depends on the multiplication rate of the parasite.

Detection of viruses in nasal swab samples from horses with acute, febrile, respiratory disease using virus isolation, polymerase chain reaction and serology

To examine the association of viruses with acute febrile respiratory disease in horses. Design Nasal swab and serum samples were collected from 20 horses with acute febrile upper respiratory disease that was clinically assessed to have a viral origin. Each of the samples was inoculated onto equine fetal kidney, RK13 and Vero cell cultures, and viral nucleic acid was extracted for polymerase chain reaction (PCR) or reverse transcription PCR. PCR primers were designed to amplify nucleic acid from viruses known to cause or be associated with acute febrile respiratory disease in horses in Australia. A type specific ELISA was used to measure equine herpesvirus (EHV1 and EHV4) antibody, and serum neutralisation assays were used to measure equine rhinitis A virus (ERAV) and equine rhinitis B virus 1 and 2 (ERBV1 and ERBV2) antibody titres in serum samples. Virus was isolated from 4 of 20 nasal swab samples. There were three isolations of EHV4 and one of ERBV2. By PCR, virus was identified in the nasal swab samples of 12 of the 20 horses. Of the 12 horses [corrected] that were positive, 17 viruses were detected as follows: there was [corrected] one triple positive (EHV4, EHV2, and EHV5), three double positives (EHV4, ERBV and EHV5, ERBV (2 horses)) and 8 [corrected] single positives (EHV4 (2 horses), EHV5 (3 horses) and ERBV (3 [corrected] horses). By virus isolation and PCR, 17 viruses were identified in nasal swab samples from 12 of 20 horses that had acute febrile respiratory disease consistent with a diagnosis of virus infection. Initial PCR identification and subsequent virus isolation led to the isolation of ERBV2 for the first time in Australia and the second time anywhere of ERBV2.

Clinical, humoral, and pathologic findings in adult alpacas with experimentally induced Corynebacterium pseudotuberculosis infection

To experimentally infect adult alpacas by ID inoculation of Corynebacterium pseudotuberculosis, follow the clinical and pathologic course of disease, and study the humoral response to infection. 13 adult alpacas. 9 alpacas were inoculated with 1.1 X 10(6) CFUs of C pseudotuberculosis from llama (n = 4) or alpaca (5) origin, and 4 alpacas were sham inoculated as controls. Alpacas were clinically observed after inoculation and euthanatized on days 16, 58, 93, or 128 after inoculation; necropsy examination and histologic evaluation were performed. An indirect ELISA, which made use of the C pseudotuberculosis cell wall as the antigen, was used to measure antibody titers in serum samples. Alpacas had a persistent febrile response, a local severe inflammatory response, and leucocytosis (> 30 X 10(3) WBCs/microL). Internal abscesses that localized mainly in the renal lymph node were observed. Corynebacterium pseudotuberculosis was recovered from the inoculation site 1 week after inoculation and from internal abscesses at 58 days after inoculation. Initial lesions were typical pyogranulomas with central caseous necrosis, whereas later lesions consisted of connective tissue, mononuclear cells, abundant neutrophils, and liquefactive necrosis. Infected alpacas had detectable serum antibody titers starting on day 16 that persisted until day 93 after inoculation. Shaminoculated alpacas did not develop serum antibody titers, clinical signs of infection, or lesions. Alpacas inoculated with C pseudotuberculosis developed abscesses at the inoculation site and internally in the renal lymph nodes, without lung lesions. Corynebacterium pseudotuberculosis isolates from llama and alpaca origin were found to be pathogenically indistinct.

Standardization of the assay method for anti-A/B antibody titers and its problems

Introduction of effective treatments such as (double filtration) plasmapheresis, splenectomy, intravenous immunoglobulin and potent immunosuppressive agents including anti-CD20 monoclonal antibody, has greatly improved graft outcome in ABO-incompatible transplantation. The measurement of anti-blood group A/B antibody titers is considered to be important, because it would provide useful information on prognosis and indication of transplantation, selection of treatments and prediction of acute rejection. Isohemagglutinin assay in serial-doubling dilutions of serum sample by tube test has been basically used in most institutions. However, detailed assay procedure seemed to vary between institutions. Furthermore, the determination by visual observation might cause a problem of inter-examiner variability. The Japanese ABO-Incompatible Transplantation Committee has conducted a series of nationwide surveys to assess the current status of inter-institutional variation in measurement of anti-A/B antibody titer. In the first survey, serum samples from 6 healthy volunteers with blood groups A ( n = 2), B ( n = 2) and O ( n = 2) were sent to 29 institutions where ABO-incompatible kidney or liver transplantation has been performed. Anti-A/B antibody titer was measured by its own protocol. Inter-institutional difference of measured values reached to 32-fold and 256-fold in IgM and IgG, respectively. As this variability was too wide for detailed analysis, the provisional standard protocol for the assay was determined according to the result of questionnaire survey. Next, 38 and 40 institutions participated in the second (in 2004) and the third survey (in 2005), respectively. Anti-A/B antibody titers of serum samples were measured by the standard procedure. Analysis of survey revealed that intra-institutional variation was reduced to below 8-fold (mostly 2- to 4-fold), although several institutions still needed some guidance. Standardization of the assay procedure and quality control survey would be necessary to improve the accuracy of measurement. Significance of measurement of anti-A/B antibody titer in ABO-incompatible transplantation should be discussed after the result of multi-center study is obtained.

Neutralizing Antibodies against Feline Parvoviruses in Nondomestic Felids Inoculated with Commercial Inactivated Polyvalent Vaccines

The virus neutralization (VN) antibody titers of serum samples from 18 individuals representing 8 carnivore species vaccinated with commercial polyvalent vaccines optimized for domestic cats containing inactivated feline panleukopenia virus (FPLV) were evaluated against canine parvovirus type 2 (CPV2). In addition, the titers among 5 individuals from 4 carnivore were evaluated against antigenic variants of feline parvoviruses; FPLV, CPV2, CPV2a, CPV2b, CPV2c, mink enteritis virus type 1 (MEV1) and MEV2. The polyvalent vaccines induced cross-reactive VN titers against antigenic variants of feline parvoviruses in nondomestic felids. However, we observed very low cross-reactive VN antibody in lions and Siberian tigers, therefore we should pay attention to CPV infections in these animals even if they were vaccinated with inactivated FPLV vaccines.