Determination Of Antibody Titers Research Articles

STANDARDIZATION OF EFFECTIVE DOSE OF FOWL CHOLERA VACCINE IN PIGEON IN BANGLADESH

An experiment was conducted to determine the effective dose of formalin killed (FK) fowl cholera (FC) vaccines prepared with virulent avian Pasteurella multocida (PM 38) serotype 1 (X-73) collected from the laboratory of the Department of Microbiology and Hygiene, BAU, Mymensingh. To determine the effective dose of vaccine, 7 weeks old 30 pigeons were immunized and each group consists of 5 birds. The groups are represented by A, B, C, D, E and F. The birds belonging to groups (A-E) were vaccinated with different doses of vaccine, after two weeks of first, second immunization and challenge experiment, blood was collected from all vaccinated birds, and serum was analyzed to determine antibody titer against P. multocida by passive hemagglutination test (PHA). The PHA titer after two weeks of first vaccination were 16±3.92, 17.6±3.92, 25.6±3.92, 32±8.76, 35.2±7.84 of group A,B,C,D and E, respectively at the dose of 0.2ml (0.26×108 CFU)/birds, 0.4ml (0.5×108 CFU)/birds, 0.8 ml (1.04×108 CFU)/birds, 1ml (1.3×108 CFU)/birds, respectively. The PHA titer of prevaccination and control birds was <4. The PHA titer after 2 weeks of second vaccination or boostering were 32±8.76, 35.2±7.84, 44.8±7.84, 57.6±18.66, 70.4±15.68, of group A,B,C,D and E, respectively. After 2 weeks of challenge infection, the mean PHA titer were 44.8±7.84, 51.2±7.84, 70.4±15.68, 102.4±15.68 and 140.8±31.34 of group A,B,C,D and E, respectively. In this experiment, the antibody titer of the vaccinated pigeons with 0.4, 0.6, 0.8 and 1ml per bird via intramuscular route were higher than that of the pigeons vaccinated with 0.4ml/bird, 0.6ml/bird, 0.8ml/bird and 1ml/bird were satisfactory in terms of protective potential against P. multocida. For prevention and control of avian pasteurellosis 0.4ml to o.6ml (0.52×108 CFU to 0.78×108 CFU)/birds of vaccine may be used instead of 1ml (1.3×108 CFU)/birds for better immunization of pigeon against fowl cholera infection.

Supplementing dietary rosemary (Rosmarinus officinalis L.) powder and vitamin E in broiler chickens: evaluation of humoral immune response, lymphoid organs, and blood proteins.

The aim of the present study was to evaluate the effect rosemary (Rosmarinus officinalis L.) powder (RP) and vitamin E (VE) at different levels on humoral immunity of broilers during a 42-day production cycle. A total of 270 1-day-old male chicks were assigned to nine groups with three replicates of ten birds each, and diets were supplemented with 0, 0.5, or 1.0% RP and 0, 100, or 200mg/kg VE, respectively. Commercial-inactivated vaccines against avian influenza (AI) and Newcastle disease (ND) viruses, and living infectious bronchitis virus (IBV) vaccine were administered by spray method. Sheep red blood cells (SRBC) were administered subcutaneously. Blood samples were collected from birds 1week after each vaccination to determine antibody titers. At the 42nd day, blood samples were also assessed for globulin level, and lymphoid tissues (thymus, spleen, and bursa) were weighed. Neither antibody titers against viruses nor lymphoid tissues weight were affected by RP and/or VE (P > 0.05) treatments. However, broilers supplemented with 0mg/kg of VE had lower antibody titers against SRBC than those fed 100mg/kg of VE (P < 0.05) at the 24th day. A significant RP × VE interaction effect (P < 0.05) on plasma globulin level was observed. The findings of our study suggest that dietary RP and VE additives can interact and modulate the humoral immunity of broilers, but not sufficiently to improve antibody titers against specific virus during a 42-day production cycle.

An Optimized Hemagglutination Inhibition (HI) Assay to Quantify Influenza-specific Antibody Titers

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody production pre- and post-vaccination is required to understand vaccine-induced immunity. This article describes a reliable point-by-point protocol to determine influenza-specific antibody titers. The first protocol describes a method to specify the antigen amounts required for hemagglutination, which standardizes the concentrations for subsequent usage in the second protocol (hemagglutination assay, HA assay). The second protocol describes the quantification of influenza-specific antibody titers against different viral strains by using a serial dilution of human serum or cell culture supernatants (hemagglutination inhibition assay, HI assay). As an applied example, we show the antibody response of a healthy cohort, which received a trivalent inactivated influenza vaccine. Additionally, the cross-reactivity between the different influenza viruses is shown and methods to minimize cross-reactivity by using different types of animal red blood cells (RBCs) are explained. The discussion highlights advantages and disadvantages of the presented assays and how the determination of influenza-specific antibody titers can improve the understanding of vaccine-related immunity.

An Optimized Hemagglutination Inhibition (HI) Assay to Quantify Influenza-specific Antibody Titers.

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody production pre- and post-vaccination is required to understand vaccine-induced immunity. This article describes a reliable point-by-point protocol to determine influenza-specific antibody titers. The first protocol describes a method to specify the antigen amounts required for hemagglutination, which standardizes the concentrations for subsequent usage in the second protocol (hemagglutination assay, HA assay). The second protocol describes the quantification of influenza-specific antibody titers against different viral strains by using a serial dilution of human serum or cell culture supernatants (hemagglutination inhibition assay, HI assay). As an applied example, we show the antibody response of a healthy cohort, which received a trivalent inactivated influenza vaccine. Additionally, the cross-reactivity between the different influenza viruses is shown and methods to minimize cross-reactivity by using different types of animal red blood cells (RBCs) are explained. The discussion highlights advantages and disadvantages of the presented assays and how the determination of influenza-specific antibody titers can improve the understanding of vaccine-related immunity.

Abstract 3228: Development and characterization of in vitro assays to detect and quantitate tubulysin B hydrazide in biological samples

Abstract EC1456 is a folate-targeted small molecule drug conjugate (SMDC) of tubulysin B hydrazide (TubBH) that is currently in Phase I clinical studies for treatment of patients with folate receptor (FR)-positive tumors. The ability to detect, quantitate, and localize drug in tissues and biological fluids is particularly important for the characterization of its biodistribution and pharmacodynamic properties. To aid in the biological characterization of our targeted TubBH conjugates, our lab had a rabbit polyclonal antibody produced against TubBH for use in various assays. An IgG-purified fraction of antiserum was used to develop competitive ELISA, immunohistochemical, and flow cytometry methods for analysis of biological samples, such as xenograft tumors from animals dosed with drug. Before assay development, a direct ELISA was performed to determine antibody titer and specificity. The TubBH antibody showed excellent binding to immobilized TubBH, whereas a rabbit IgG negative control antibody had minimal binding. Next, a competitive ELISA was developed in which soluble TubBH competed for antibody binding to immobilized antigen. Multiple experimental parameters were optimized, including coating antigen concentration, antibody dilution, and sample matrix extraction method. Using the optimized conditions, the resulting standard curves for TubBH in acetonitrile-extracted KB tumor sample matrix showed good sensitivity and dose-response. It was determined that, in addition to untargeted TubBH, the antibody could also be used to quantitate intact EC1456, as well as a metabolite of the drug lacking the hydrazide. The utility of the antibody was further demonstrated utilizing a PSMA-targeted version of TubBH. Once optimized, the ELISA method was used to analyze FR-positive KB and FR-negative A549 xenograft tumor homogenates from animals dosed with either EC1456 or untargeted TubBH. The levels of TubBH were higher 4h-post EC1456 dose in the FR-positive KB tumors compared to the FR-negative A549 tumors; however, levels were similar after 24h. Moreover, untargeted TubBH resulted in lower tumor concentrations of the drug compared to EC1456 in KB tumors. An immunohistochemical method developed using the antibody showed strong, focal, cytoplasmic staining in FFPE tumor sections of KB tumors from animals dosed with EC1456, while no staining was observed in tumors from undosed animals. Finally, the antibody was successfully used to detect cell surface FR-bound EC1456 on fixed and unfixed KB cells in vitro by flow cytometry. This experiment confirmed that the antibody is able to recognize the conjugate even when it is situated in the FR binding pocket. Collectively, these results show that this antibody against TubBH can be used as a powerful tool for analysis of tumor drug uptake for folate-targeted TubBH in xenograft models and could hold great potential for use in patient sample analysis as well. Citation Format: Nikki L. Parker, Jonathan M. Shillingford, Melissa Nelson, Joseph A. Reddy, Christopher P. Leamon. Development and characterization of in vitro assays to detect and quantitate tubulysin B hydrazide in biological samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3228. doi:10.1158/1538-7445.AM2017-3228

The methodology of creating a vaccine against candidiasis

Candidiasis is one of the most common diseases of our time. For many years to treat candidiasis the same antifungal drugs, to which the fungi gradually develop resistance, have been used. One of the ways to solve the problem of effective treatment of candidiasis is to use vaccines, which have a number of significant advantages compared to other antifungal drugs. Currently, there are no vaccines against candidiasis at the pharmaceutical market.Aim. To substantiate the general concept for creation of a new vaccine based on Candida fungi.Materials and methods. Development of a new vaccine should be combined on a common methodological approach to pharmaceutical development taking into account the requirements for a dosage form. In order to determine the general concept for creation of a new vaccine the following areas of research have been outlined: development of a new vaccine based on inactivated cells of Candida genus fungi and creation of a new vaccine for the renewal of proteins and polysaccharides of fungi of Candida genus fungi.Results. To provide the effectiveness and safety of the vaccine all pharmaceutical and technological factors that affect the vaccine should be theoretically and experimentally substantiated. They include the nature and the amount of active substances, technological parameters for preparation and mixing of active substances, the physicochemical and biological properties of active substances and excipients, equipment. In order to provide the therapeutic effectiveness of the vaccine the biopharmaceutical studies to determine antibody titers and efficacy in the prevention and treatment of candidal infection were conducted.Conclusions. Thus, on the basis of the universal methodological approach to pharmaceutical development the methodology has been substantiated; the plan for experimental studies on creating the solution of the vaccine for injection to prevent and treat candidiasis on the basis of fungal cells of Candida genus has been developed.

Microarray profile of the humoral immune response to influenza vaccination in breast cancer patients treated with chemotherapy

BackgroundPatients treated with chemotherapy have an impaired response to influenza virus vaccination compared to healthy controls. Little is known about the broadness of the antibody response in these patients. MethodsBreast cancer patients on FEC (5-fluorouracil, epirubicin and cyclophosphamide) chemotherapy regimens were vaccinated with influenza virus vaccine. Sera were obtained before and three weeks after vaccination. In addition to the determination of virus-specific antibody titres by hemagglutination inhibition assay, the broadness of the response was assessed by the use of a protein microarray and baseline titres were compared with an age-matched reference group. ResultsWe included 38 breast cancer patients and found a wide variety in serum antibody response after vaccination. Patients with a history of influenza vaccination had higher pre-vaccination titres, which were comparable to the reference group.Increasing number of cycles of chemotherapy did not have a negative effect on influenza array antibody levels, nor on the HI antibody response. ConclusionsOverall there was a broad serum antibody response to the influenza virus vaccine in patients treated with chemotherapy for breast cancer.

Serologic survey for antibodies against three genotypes of bovine parainfluenza 3 virus in unvaccinated ungulates in Alabama.

OBJECTIVE To determine titers of serum antibodies against 3 genotypes of bovine parainfluenza 3 virus (BPI3V) in unvaccinated ungulates in Alabama. ANIMALS 62 cattle, goats, and New World camelids from 5 distinct herds and 21 captured white-tailed deer. PROCEDURES Serum samples were obtained from all animals for determination of anti-BPI3V antibody titers, which were measured by virus neutralization assays that used indicator (reference) viruses from each of the 3 BPI3V genotypes (BPI3V-A, BPI3V-B, and BPI3V-C). The reference strains were recent clinical isolates from US cattle. Each sample was assayed in triplicate for each genotype. Animals with a mean antibody titer ≤ 2 for a particular genotype were considered seronegative for that genotype. RESULTS Animals seropositive for antibodies against BPI3V were identified in 2 of 3 groups of cattle and the group of New World camelids. The geometric mean antibody titer against BPI3V-B was significantly greater than that for BPI3V-A and BPI3V-C in all 3 groups. All goats, captive white-tailed deer, and cattle in the third cattle group were seronegative for all 3 genotypes of the virus. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that BPI3V-A may no longer be the predominant genotype circulating among ungulates in Alabama. This may be clinically relevant because BPI3V is frequently involved in the pathogenesis of bovine respiratory disease complex, current vaccines contain antigens against BPI3V-A only, and the extent of cross-protection among antibodies against the various BPI3V genotypes is unknown.

Systematic Comparison of IgM and IgG ABO Antibody Titers by Using Tube and Gel Card Techniques and its Relevance for ABO-Incompatible Kidney Transplantation.

Although the determination of the ABO antibody titers is necessary for the decision-making in ABOincompatible (ABOi) kidney transplantations, various methods for the determination of the ABO antibody titers are being used. However, the absence of uniform standards makes their comparability far more difficult. Two of the most commonly used methods are the tube method and the gel card method. In this study, we systematically investigate to what extent these two methods affect the result of ABO antibody titers. ABO antibodies were determined from plasmas of 90 donors (30 individuals each with blood group A, B, and O). Seven further donors with blood group A, B, and AB provided erythrocytes for the testing. A total of 360 ABO antibody titers were determined; 180 tests for each method, each with 90 determinations of immunoglobulin G (IgG) and immunoglobulin M (IgM) antibody titers. In addition, we also made a differentiation by blood groups to find out if and to what extent the blood groups have an impact on the results. Our analysis shows that the choice of method has a highly significant (p < 0.0001) impact on the titer level of the ABO antibodies. The median values of ABO antibody titers determined by using the gel card method are two titer steps lower than the titers, which are determined when using the tube method. Moreover, our data shows that there are major differences in the ABO antibody titer level among the blood groups, regardless of the choice of methods. We consider changing to the gel card method for determining the ABO antibody titers as a simple and effective way to achieve a standardized and uniform method. Here, too, the clinicians should be provided with sufficient information by the laboratories, in order to draw the right consequence from this change, while considering all the relevant data. As a consequence of this study, the transplant center of the University of Hamburg-Eppendorf paired a change from tube to gel card regarding the ABO antibody titer determination of ABOi kidney transplantations with an intensification of the preoperative target titer from ≤ 1:8 to ≤ 1:4.

Effects of Thymoquinone on Interleukin-1 and Interferon Gamma Gene Expression and Antibody Titers against Newcastle Disease in Broiler Chickens under Oxidative Stress

ABSTRACT An experiment was conducted to determine the effects of the dietary inclusion of different levels of thymoquinone (TQ) of broilers subjected to oxidative stress or not on the antibody titers against Newcastle disease and on the gene expression of interleukine-1 and interferon gamma. A total of 320 one-day-old broilers was randomly assigned to eight treatments with four replicates of 10 birds each, in a 4 × 2 factorial arrangement, consisting of four thymoquinone (TQ) levels (0, 5, 8, or 11 mg/kg body weight) and two levels tert-butyl hydroperoxide (t-BHP) injection (0 or 0.02 mmol/kg of body weight). Blood samples were collected from two birds per replicate to determine antibody titers against Newcastle disease. At the end of experiment, two birds per replicate were randomly selected, sacrificed and their spleens were collected to evaluate the genes expressioninterleukin-1 and interferon gamma (p<0.05). The dietary inclusion of TQ of broilers subjected or not oxidative stress increased antibody production against Newcastle disease (p<0.05). Both individual and combined dietary inclusion of t-BHP and TQ promote the differentiation and proliferation of spleen cells and the gene expression of interleukin-1 and interferon gamma (p<0.05).

Aspergillusserology: Have we arrived yet?

Aspergillosis presents in various clinical forms, among them chronic pulmonary aspergillosis, which is a spectrum of disease entities including aspergilloma, chronic cavitary pulmonary aspergillosis, and chronic fibrosing pulmonary aspergillosis. Aspergillus also contributes to fungal allergy and sensitization. Analysis of the immune response to Aspergillus and its antigens is an integral part of the diagnosis of these diseases. Over the past half century, the techniques used to determine antibody titers have evolved from testing for precipitating and agglutinating antibodies by agar gel double diffusion and immunolectrophoresis to enzyme-linked immunosorbent assays using recombinant proteins as capture antigens. A resurgence of interest in the detection of immunoglobulins, primarily Aspergillus-specific IgG, has hinted at the possibility of distinguishing between colonization and invasion in immunocompromised patients with invasive aspergillosis. Even though there appears to be a greater degree of discrimination between the clinical forms of aspergillosis there is still a long way to travel. This review presents illustrative examples of where new diagnostic platforms and technologies have been applied to this intriguing spectrum of diseases.

Effects of injectable trace minerals on humoral and cell-mediated immune responses to Bovine viral diarrhea virus, Bovine herpes virus 1 and Bovine respiratory syncytial virus following administration of a modified-live virus vaccine in dairy calves

Our objective was to evaluate the effect of an injectable trace mineral (ITM) supplement containing zinc, manganese, selenium, and copper on the humoral and cell mediated immune (CMI) responses to vaccine antigens in dairy calves receiving a modified-live viral (MLV) vaccine containing BVDV, BHV1, PI3V and BRSV. A total of 30 dairy calves (3.5 months of age) were administered a priming dose of the MLV vaccine containing BHV1, BVDV1 & 2, BRSV, PI3V, and an attenuated-live Mannheimia-Pasteurella bacterin subcutaneously (SQ). Calves were randomly assigned to 1 of 2 groups: (1) administration of ITM SQ (ITM, n=15) or (2) injection of sterile saline SQ (Control; n=15). Three weeks later, calves received a booster of the same vaccine combination SQ, and a second administration of ITM, or sterile saline, according to the treatment group. Blood samples were collected on days 0, 7, 14, 21, 28, 42, 56, and 90 post-vaccination for determination of antibody titer, viral recall antigen-induced IFN-γ production, and viral antigen-induced proliferation by peripheral blood mononuclear cells (PBMC). Administration of ITM concurrently with MLV vaccination resulted in higher antibody titers to BVDV1 on day 28 after priming vaccination compared to the control group (P=0.03). Calves treated with ITM showed an earlier enhancement in PBMC proliferation to BVDV1 following vaccination compared to the control group. Proliferation of PBMC after BVDV stimulation tended to be higher on day 14 after priming vaccination in calves treated with ITM than in the control group (P=0.08). Calves that received ITM showed higher PBMC proliferation to BRSV stimulation on day 7 after priming vaccination compared to the control group (P=0.01). Moreover, calves in the ITM group also had an enhanced production IFN-γ by PBMC after stimulation with BRSV on day 21 after priming vaccination compared to day 0 (P<0.01). In conclusion, administration of ITM concurrently with MLV vaccination in dairy calves resulted in increased antibody titer to BVDV1, and greater PBMC proliferation to BVDV1 and BRSV recall stimulation compared to the control group, suggesting that ITM might represent a promising tool to enhance the humoral and CMI responses to MLV vaccines in cattle.

Use of three-dimensional accelerometers to evaluate behavioral changes in cattle experimentally infected with bovine viral diarrhea virus.

OBJECTIVE To assess the use of 3-D accelerometers to evaluate behavioral changes in cattle experimentally infected with a low-virulent strain of bovine viral diarrhea virus (BVDV). ANIMALS 20 beef steers (mean weight, 238 kg). PROCEDURES Calves were allocated to a BVDV (n = 10) or control (10) group. On day 0, calves in the BVDV group were inoculated with a low-virulent strain of BVDV (4 × 10(6) TCID50, intranasally), and calves in the control group were sham inoculated with BVDV-free medium (4 mL; intranasally). An accelerometer was affixed to the right hind limb of each calf on day -7 to record activity (lying, walking, and standing) continuously until 35 days after inoculation. Baseline was defined as days -7 to -1. Blood samples were collected at predetermined times for CBC, serum biochemical analysis, virus isolation, and determination of anti-BVDV antibody titers. RESULTS All calves in the BVDV group developed viremia and anti-BVDV antibodies but developed only subclinical or mild disease. Calves in the control group did not develop viremia or anti-BVDV antibodies. Mean time allocated to each activity did not differ significantly between the BVDV and control groups on any day except day 8, when calves in the BVDV group spent less time standing than the calves in the control group. Following inoculation, calves in both groups tended to spend more time lying and less time walking and standing than they did during baseline. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that behavioral data obtained by accelerometers could not distinguish calves subclinically infected with BVDV from healthy control calves. However, subtle changes in the behavior of the BVDV-infected calves were detected and warrant further investigation.

Development of Experimental Oil based Inactivated HS Vaccine from field Isolates of Pasteurella multocida from Cattle in Bangladesh

The present research work was conducted to developed oil based inactivated HS vaccine from fields isolates of Pasteurella multocida from cattle in Bangladesh and determination of antibody titers following vaccination in bovines. For this purpose, a total of 26 samples were collected where 16 blood samples were from Bogra and 6 blood samples from Mymensingh in clinical cases and 4 morbid materials such as lung, liver, and heart were collected from suspected cases of hemorrhagic septicemia (HS) in cattle of Rajshahi. The samples were processed for demonstration of bipolar organisms and isolation of Pasteurella multocida. There were 9 samples were isolated as P. multocida in which 3 samples from Bogra, 2 samples from Mymensingh and 4 samples from Rajshahi. The isolates were identified by cultural and morphological characteristics, biochemical characteristics, serological and pathogenicity tests. The dense culture of the organism was achieved in a blood agar by incubation. The formalin inactivated HS vaccines (oil adjuvanated) was prepared and administered in cattle at the dose rate of 2ml (1.5 OD600 = ≥7.5×108 CFU/ml) through subcutaneous (SC) route in each selected Group (A, and B) in the field of Gouripur Upazilla, Mymensingh and Nokla Upazilla, Sherpur respectively. Blood was collected from each group A and B before and after 28 days of primary vaccination and pre-vaccinated sera was kept as control. The mean PHA titers were 134.86, 169.14 and <4±0.00 in Group A and B and control group respectively. The PHA titers were analyzed by t-test to determine the immune level of vaccinated cattle. The vaccine produced better immune response in post vaccinated groups of cattle as compared to prevaccinated groups of cattle. So, it may be concluded that oil based inactivated HS vaccine induced high level of passive haemagglutinating (PHA) antibodies in the vaccinated cattle.

Automated Image Analysis for Determination of Antibody Titers Against Occupational Bacterial Antigens Using Indirect Immunofluorescence.

Employees who are exposed to high concentrations of microorganisms in bioaerosols frequently suffer from respiratory disorders. However, etiology and in particular potential roles of microorganisms in pathogenesis still need to be elucidated. Thus, determination of employees' antibody titers against specific occupational microbial antigens may lead to identification of potentially harmful species. Since indirect immunofluorescence (IIF) is easy to implement, we used this technique to analyze immunoreactions in human sera. In order to address disadvantageous inter-observer variations as well as the absence of quantifiable fluorescence data in conventional titer determination by eye, we specifically developed a software tool for automated image analysis. The 'Fluorolyzer' software is able to reliably quantify fluorescence intensities of antibody-bound bacterial cells on digital images. Subsequently, fluorescence values of single cells have been used to calculate non-discrete IgG titers. We tested this approach on multiple bacterial workplace isolates and determined titers in sera from 20 volunteers. Furthermore, we compared image-based results with the conventional manual readout and found significant correlation as well as statistically confirmed reproducibility. In conclusion, we successfully employed 'Fluorolyzer' for determination of titers against various bacterial species and demonstrated its applicability as a useful tool for reliable and efficient analysis of immune response toward occupational exposure to bioaerosols.

Preparation of Inactivated Trivalent FMD Vaccine and Determination of Antibody Titre in Vaccinated Cattle

Aims: This research work was conducted for isolation and molecular detection of Foot and Mouth Disease (FMD) virus from the field samples, development of inactivated trivalent FMD vaccine, and determination of antibody titer in vaccinated cattle. Methodology: A total of 10 samples (tongue epithelium) were collected from FMD affected cattle from Gazipur, Mymensingh, and Pabna districts of Bangladesh during May 2014. Inoculum was prepared from the sample and the associated FMDV was propagated in BHK-21 cell lines. Besides, viral RNA was extracted for molecular detection (RT-PCR) of the serotypes involved. The isolated serotypes were used as seed virus in preparation of binary ethyleneimine (BEI) inactivated trivalent FMD vaccine using saponin and oil adjuvants. For the determination of antibody production in Original Research Article Chowdhury et al.; IJTDH, 16(2): 1-8, 2016; Article no.IJTDH.25870 2 response to our trivalent FMD vaccine, a total of 25 cattle aging 11-22 months were used. Sera of vaccinated cattle were collected on day 0, 21, 35, 49, 63. The sera were subjected for the determination of antibody level using enzyme-linked immunosorbent assay (ELISA). The titer values were statistically analyzed using one-way ANOVA to see immune response against the serotypes. Results: Three serotypes of FMDV were detected; these were FMD serotype-O, A and Asia-1, of which serotype-O was mostly prevalent, followed by serotype-A and Asia-1. The highest mean antibody titer was found on day 63 in all serotypes. Sixteen cattle (80%) out of 20 vaccinated cattle obtained protective antibody titer. The titer values of the vaccinated cattle were statistically significant against O, A and Asia-1 serotypes. Conclusion: FMD serotype-O, A, and Aisa-1 are prevailing in Bangladesh. A trivalent inactivated FMD vaccine has been prepared successfully using circulating virus of Bangladesh. The vaccine can be used to combat FMD in Bangladesh.

Comparable immune responsiveness but increased reactogenicity after subcutaneous versus intramuscular administration of tick borne encephalitis (TBE) vaccine

Evaluation of safety, immunogenicity and efficacy of vaccines during licensing studies is performed in relation to the selected vaccination route. For most adjuvanted vaccines, such as the TBE vaccine FSME-IMMUN, only intramuscular (i.m.) administration is licensed. Yet in certain situations, either because of medical indications, accidental application or due to a lack of sufficient muscular tissue, the vaccine might rather be applied subcutaneously (s.c.). With respect to the TBE vaccine there are currently however no data to support the use of the subcutaneous route of vaccination.In order to compare the reactogenicity and immune responsiveness upon i.m. and s.c. TBE vaccination 116 (58 females and 58 males) participants with a documented primary TBE vaccination course were randomized to receive either an i.m. or s.c. booster. Venous blood was collected before, 7 days, 1 month and 6 months after vaccination to determine antibody titer profiles. PBMC were isolated prior to and 7 days after booster to analyze lymphocyte subpopulations and cytokine production upon antigen restimulation. Subjects were monitored for the occurrence of side effects for 7 days post vaccination.Comparable levels of TBE specific neutralizing antibodies were induced after s.c. and i.m. vaccination. At the cellular level, IL-2, IFN gamma and IL-10 levels did not significantly differ using either route of vaccination and the distribution of T cell subsets was comparable along with a relative decrease of regulatory T-cells after both ways of administration.In contrast to the immunogenicity analyses, the data from safety diaries revealed a significantly higher rate of local, but not of systemic reactions after s.c. administration. In conclusion, this study demonstrates that both routes lead to comparable immune responses to the TBE antigen. The higher rate and intensity of local reactions, particularly among women, after s.c. vaccination however needs to be addressed during counseling.

Urinary shedding of leptospires and presence of Leptospira antibodies in healthy dogs from Upper Bavaria.

Leptospirosis is classified as a re-emerging zoonotic disease with global impor- tance. The aim of this study was to determine urinary shedding of leptospires in healthy dogs and to identify the shedded leptospire species. Furthermore, antibody presence against leptospires was evaluated. In a prospective study urine samples of 200 healthy dogs from Upper Bavaria were randomly collected and evaluated by real-time polymerase chain reaction (PCR) specific for the lipL32 gene of pathogenic Leptospira (L) spp. Positive samples were further character- ized via multilocus sequence typing (MLST) to identify the Leptospira species. Microagglutination test (MAT) was performed to determine serum antibody titers. Three of 200 urine samples were found to be PCR-positive resulting in a urinary shedding prevalence of 1.5% (95% confidence interval 0.3-4.5%). All three dogs had been vaccinated before with a bivalent vaccine, covering the serogroups Canicola and lcterohaemorrhagiae. One dog shed leptospires of the species L. borgpetersenii, and two of the species L. interrogans. Of all dogs, 17.0% had antibody titers ≥ 1:100, and 3.5% titers ≥ 1:400 to serovars of non-vaccinal sero- groups. Healthy dogs that shed leptospires represent a possible risk for humans and other animals. The study emphasizes the importance of general hygiene measures in veterinary practice while handling urine of all dogs, and the use of vaccines that protect against a broader range of serogroups and that prevent urinary shedding.

Isolation of Pasteurella multocida from chickens, preparation of formalin killed fowl cholera vaccine, and determination of efficacy in experimental chickens

Objectives: The objectives of this study were to isolate and identify Pasteurella multocida from fowl cholera (FC) suspected chicken, and to prepare and efficacy determination of formalin killed fowl cholera vaccine using the isolated P. multocida strain. Materials and methods: A total of five suspected dead chickens were collected from Brothers Poultry Farm located at Gazipur district, Bangladesh. The samples were processed and the P. multocida was isolated through conventional bacteriological techniques, were finally confirmed by polymerase chain reaction using P. multocida specific primers targeting cap gene. The P. multocida isolate was used to develop a formalin killed fowl cholera vaccine. The efficacy of the newly prepared vaccine was determined in Starcross-579 chickens (n=30) aging 15 weeks either by injecting 1 mL (group-A; n=10) or 0.5 mL (group-B; n=10) vaccine containing approximately 3.2x108 CFU/mL P. multocida organism; 10 birds were kept as unvaccinated control. The sera from the vaccinated and control birds were collected and were subjected for antibody titre determination by enzyme-linked immunosorbent assay (ELISA). Finally the vaccinated birds were challenged using virulent strains of P. multocida to confer the protection against FC. Results: P. multocida could be isolated from both the samples. The formalin killed vaccine prepared from the isolated bacteria was subjected for the determination of antibody titre in chicken, and found that the antibody titres in the birds of group A and group B were 4.513 and 4.07 respectively after primary vaccination, and 4.893 and 4.37 respectively after booster vaccination. Most of the vaccinated birds were found to be survived after challenging with virulent strain of P. multocida. Conclusion: It is concluded that the causal agent of FC (P. multocida) was successfully isolated from FC affected dead chickens. The prepared formalin killed fowl cholera vaccine induces protective immune response and conferred protection against challenge infection caused by the virulent strain of P. multocida.

The Effectiveness of Vaccination in Sickly Children with Disorders of the Central Nervous System

Data on the current vaccination process have in sickly children with disorders of the central nervous system, and grafted lifeless live vaccines against diphtheria, measles and mumps.The levele of intercurrent diseases in every group was 33,3%. The post-vaccination period in 73% vaccination to diphtheria are asymptomatic (and after measles mumps vaccination in 86%). The post-vaccination reactions were in 50% illness children. The antibody titers seronegative was not identified. Children with epilepsia need to determine antibody titers sixs months after vaccination against measles and mumps to decide on supplementary immunization.