Assessment using ELISA of the herd immunity levels induced in cattle by foot-and-mouth disease oil vaccines

The development of a liquid-phase blocking sandwich ELISA (LPBE) to measure antibodies (Ab) produced in cattle with the O, A and C foot-and-mouth disease virus (FMDV) types of commercial vaccines used in Argentina is described. The test was specific: 99% of na?ve cattle sera (n = 130) gave titres below log10 = 1.2, and none had a titre above log10 = 1.5. Comparative studies with serum neutralization test (SNT) using sera from cattle which received one or more vaccine doses is reported. The overall rank correlation coefficient (Spearman's rho, rs) between SNT and LPBE were highly significant (rs > 0.67, P < 0.0001) for all vaccine strains. LBPE Ab titres on sera collected 90 days post vaccination were compared with results of cattle protection tests by applying a logistic regression. The minimum Ab titres at which 85% and 75% of the cattle were protected for each FMDV type were determined in order to interpret field Ab data in terms of protection. Application of this method allows large scale serological examinations to monitor antibody levels in vaccinated animals as an indirect indicator of the FMD control program status in the field. Its use in the evaluation of commercial batches of FMD vaccine is discussed.     

Comparison of selected canine vaccines for their ability to induce protective immunity against canine parvovirus infection

BACKGROUND AND AIMS: The ingestion of a corrosive agent including strong alkaline material causes serious caustic damage to the upper gastrointestinal tract. We describe four cases in patients who had ingested alkaline substances. MATERIAL AND METHODS: During the years 1992-1996, four patients who had ingested strong alkali were treated in the Oulu University Hospital. The patients were reviewed retrospectively. RESULTS: In one case a third-degree, in two cases a second-degree and in one case a first-degree injury developed in the oesophagus. The patient with first-degree injury was treated with repeated endoscopic dilatations and he refused any more aggressive surgical therapy. The patients with more severe injuries were operated, on all with good end results. CONCLUSION: Aggressive surgical treatment of severe corrosive injuries involving the upper gastrointestinal tract is recommended.     

DNA vaccines with single-chain Fv fused to fragment C of tetanus toxin induce protective immunity against lymphoma and myeloma

Vaccination with idiotypic protein protects against B-cell lymphoma, mainly through anti-idiotypic antibody. For use in patients, DNA vaccines containing single-chain Fv derived from tumor provide a convenient alternative vaccine delivery system. However, single-chain Fv sequence alone induces low anti-idiotypic response and poor protection against lymphoma. Fusion of the gene encoding fragment C of tetanus toxin to single-chain Fv substantially promotes the anti-idiotypic response and induces strong protection against B-cell lymphoma. The same fusion design also induces protective immunity against a surface Ig-negative myeloma. These findings indicate that fusion to a pathogen sequence allows a tumor antigen to engage diverse immune mechanisms that suppress growth. This fusion design has the added advantage of overcoming potential tolerance to tumor that may exist in patients.     

Evaluation of the therapeutic and protective efficacy of doramectin against psoroptic scabies in cattle

Arenaviruses are enveloped viruses with a genome composed of two ssRNA species, designated L and S. The arenaviruses were divided in two major groups (Old World and New World), based on serological properties and genetic data, as well as geographic distribution. A sequence alignment analysis of all reported arenavirus S RNAs yielded 17 conserved regions in addition to a reported conserved region at the end of both RNAs. The consensus sequences of these regions were used to design generalized primers suitable for RT-PCR amplification of a set of overlapping nucleotide sequence fragments comprising the complete S RNA of any arenavirus. A restriction analysis (RFLP) was designed to rapidly typify the amplified fragments. This RT-PCR-RFLP approach was tested with Old World (LCM) and New World (Junin and Tacaribe) arenaviruses. Furthermore, using this procedure the whole S RNA of a novel arenavirus isolate obtained from a rodent trapped in central Argentina, was amplified and characterized. Partial nucleotide sequence data were used for phylogenetic analyses that showed the relationships between this arenavirus and the rest of the members of the family. This relatively simple methodology will be useful both in basic studies and epidemiological survey programs.     

Observations on the use of Anaplasma centrale for immunization of cattle against anaplasmosis in Zimbabwe

To assess the safety and efficacy of consensus interferon (IFN-Con-1), 55 patients with chronic hepatitis C infection were treated with either 3, 6, 9, 12, or 15 microg IFN-Con-1 s.c. three times a week for 24 weeks, followed by 24 weeks of observation. There was a dose-response relationship with respect to the number of patients with normalized ALT concentrations or undetectable HCV RNA. At the end of the 24-week treatment period, the serum ALT had normalized in 18% of patients given the 3 microg dose and 42% of patients given the 12 microg or 15 microg doses of IFN-Con-1. At the end of the posttreatment observation period, the serum ALT was still normal in 10% of patients given the 3 microg, 6 microg, or 9 microg doses and in 50% of patients given the 15 microg dose. Also, at the end of the 24-week treatment period, 27% of patients given the 3 microg dose and 75% given the 15 microg dose had undetectable serum HCV RNA. At the end of the posttreatment observation period, the proportion of patients with undetectable HCV RNA ranged from 9% of those given the 3 microg dose to 50% of those given the 15 microg dose. Our study indicates that treatment with IFN-Con-1 appears to be safe and effective. In addition, use of 15 microg of IFN-Con-1 resulted in significantly more patients with sustained ALT normalization and absence of HCV RNA 6 months after cessation of therapy compared with treatment with lower doses of IFN-Con-1. Additional trials are underway to confirm these findings.     

Evaluation of the antibody response in pigs vaccinated against Streptococcus suis capsular type 2 using a double-antibody sandwich enzyme-linked immunosorbent assay

OBJECTIVE: We assessed the value of dynamic sequential three-dimensional Fourier transformation (3DFT) MRI in differentiating various types of small liver tumors. MATERIALS AND METHODS: Forty-seven patients with 65 liver masses < 3 cm in size (42 hepatocellular carcinomas, 11 hemangiomas, 12 metastatic tumors) were studied by 3DFT fast imaging with steady-state precession (FISP) MRI [TR(ms)/TE(ms)/flip angle (degree): 20/8/30]. The slab thickness was 21-35 mm, and there were seven partitions. The 3DFT-FISP MR images were obtained immediately after 0.1 mmol/kg of gadopentetate dimeglumine was administered intravenously over 2-3 s (early phase), 60 s after (late phase I), and 120 s after (late phase II). RESULTS: Eighty-six percent of small hepatocellular carcinomas showed hyperintense enhancement relative to the surrounding liver parenchyma and iso- or hypointense enhancement with or without capsular enhancement in the late phase. Eighty-two percent of small hemangiomas showed peripheral globular enhancement in the early phase and total hyperintense or peripheral enhancement in the late phases. Ninety-two percent of the small metastatic liver tumors showed doughnut-like ring enhancement in the early phase. CONCLUSION: By dynamic 3DFT-FISP MRI, we were able to accurately evaluate the hemodynamics and morphological findings of each type of small liver tumor.     

Present status of the use of cytokines as adjuvants with vaccines to protect against infectious diseases

Vaccine adjuvants are expected to play an important role in enhancing the immunogenicity of existing and new-generation vaccines against infectious diseases. In particular, adjuvants should direct the immune response in the most appropriate manner--furthering, for example, an expanded B-cell response, a cytotoxic T-cell response, or a T-helper 1 or 2 subset response. While some noncytokine adjuvants have exerted potent effects, their modes of action are most likely mediated by cytokines. Several cytokines have already been shown to be efficient adjuvants in animal models and/or in clinical trials. The mechanisms of cytokine function must be better understood and the techniques for the use of cytokines improved if the full potential of these substances as vaccine adjuvants is to be realized. When used to best advantage, such adjuvants enhance the immunity induced by viral, bacterial, and parasitic vaccines and thereby promote efficient protection or even cure.     

Limited breadth of the protective immunity elicited by simian immunodeficiency virus SIVmne gp160 vaccines in a combination immunization regimen

We previously reported that immunization with recombinant simian immunodeficiency virus SIVmne envelope (gp160) vaccines protected macaques against an intravenous challenge by the cloned homologous virus, E11S. In this study, we confirmed this observation and found that the vaccines were effective not only against virus grown on human T-cell lines but also against virus grown on macaque peripheral blood mononuclear cells (PBMC). The breadth of protection, however, was limited. In three experiments, 3 of 10 animals challenged with the parental uncloned SIVmne were completely protected. Of the remaining animals, three were transiently virus positive and four were persistently positive after challenge, as were 10 nonimmunized control animals. Protection was not correlated with levels of serum-neutralizing antibodies against the homologous SIVmne or a related virus, SIVmac251. To gain further insight into the protective mechanism, we analyzed nucleotide sequences in the envelope region of the uncloned challenge virus and compared them with those present in the PBMC of infected animals. The majority (85%) of the uncloned challenge virus was homologous to the molecular clone from which the vaccines were made (E11S type). The remaining 15% contained conserved changes in the V1 region (variant types). Control animals infected with this uncloned virus had different proportions of the two genotypes, whereas three of four immunized but persistently infected animals had >99% of the variant types early after infection. These results indicate that the protective immunity elicited by recombinant gp160 vaccines is restricted primarily to the homologous virus and suggest the possibility that immune responses directed to the V1 region of the envelope protein play a role in protection.     

The efficacy of recombinant vaccines against hepatitis B and the results of their use in a schedule for pediatric prophylactic inoculations

The results of field clinical trials of Russian and American yeast vaccines against hepatitis B are presented. The study revealed that both vaccines were faintly reactogenic, safe and exhibited high immunological activity. After the full course of immunization following the schedule 0-1-2 months 92.5% and 97.5% of patients receiving, respectively. Russian vaccine "Combiotech" and American vaccine "H-B-Vax II" were found to have specific antibodies. The maximum effect was registered when the vaccines were introduced according to the schedule 0-1-6 months. Seroconversions were observed in 97.5% and 100% of the vaccinees receiving the Russian and American vaccines respectively, in the latter case the highest antibody level being observed. The use of the vaccines within the prophylactic immunization schedule showed that antibodies to hepatitis B appeared in immunized children in 93-100% of cases. Seroconversion indices and the levels of antibodies to diphtheria, tetanus, poliomyelitis and measles were statistically significant and were the same in children receiving only the vaccines according to the immunization schedule and in children immunized, in addition to these vaccines, against hepatitis B.     

Induction of protective immunity against Japanese encephalitis in mice by immunization with a plasmid encoding Japanese encephalitis virus premembrane and envelope genes

A DNA vaccine plasmid containing the Japanese encephalitis (JE) virus premembrane (prM) and envelope (E) genes (designated pcDNA3JEME) was evaluated for immunogenicity and protective efficacy in mice. Two immunizations of 4-week-old female ICR mice with pcDNA3JEME by intramuscular or intradermal injections at a dose of 10 or 100 microg per mouse elicited neutralizing (NEUT) antibodies at titers of 1:10 to 1:20 (90% plaque reduction), and all immunized mice survived a challenge with 10,000 50% lethal doses of the P3 strain of JE virus. A single immunization with 100 microg of pcDNA3JEME did not elicit detectable NEUT antibodies but induced protective immunity. Spleen cells obtained from BALB/c mice immunized once with 10 or 100 microg of pcDNA3JEME contained JE virus-specific memory cytotoxic T lymphocytes (CTLs). BALB/c mice maintained detectable levels of memory B cells and CTLs for at least 6 months after one immunization with pcDNA3JEME at a dose of 100 microg. The CTLs induced in BALB/c mice immunized twice with 100 microg of pcDNA3JEME were CD8 positive and recognized mainly the envelope protein. These results indicate that pcDNA3JEME has the ability to induce a protective immune response which includes JE virus-specific antibodies and CTLs.     

Utilization of a direct sandwich ELISA kit for rapid diagnosis of adenovirus hemorrhagic cystitis in a patient with adult T-cell leukemia

We report on a case of adenovirus hemorrhagi cystitis that developed in a 49-year-old woman during intensive chemotherapy for adult T-cell leukemia. Although rapid etiologic diagnosis is essential for the effective management of viral hemorrhagic cystitis, the isolation of adenovirus from urine is often too time-cousuming. We detected the adenovirus antigen in the patient's urine using an Adenoclone direct sandwich ELISA kit (Cambridge Bio Science), which is commonly employed for the diagnosis of adenovirus conjunctivitis. Treatment consisted of intravenous vidarabine and bladder irrigation, which resulted in prompt clinical improvement. The Adenoclone kit was useful for the rapid etiologic diagnosis of adenovirus hemmorrhagic cystitis.     

Diagnosis of freemartinism in cattle: the need for clinical and cytogenic evaluation

A total of 727 blood samples from female calves born co-twin to male calves were examined cytogenetically for freemartinism between 1978 and 1992. Six hundred calves (82.5%) were determined to be freemartins, and 127 (17.5%) were determined not to be freemartins. The percentage of calves determined not to be freemartins was substantially higher than the 8% reported for an unselected population of female co-twins. We concluded that some obvious freemartins were eliminated prior to submission of samples for confirmatory cytogenetic diagnosis, and that only a small percentage of the estimated 93,000 female calves born co-twin to male calves annually are so examined. Therefore, probably a large number of female co-twins that are not truly freemartins are sold to slaughter every year. We propose that obvious freemartins be identified by use of the vaginal-length test and that the remaining clinically questionable calves be differentiated cytogenetically. This combination of procedures could prevent unnecessary economic losses and preserve important genetic material. Three animals with chromosomal anomalies were found during examination of samples for freemartinism. Cytogenetic evaluation for freemartinism thus offers the added value of simultaneous surveillance for cytogenetic aberrations in male and female cells of a sample.     

The use of a cationic liposome formulation (DOTAP) mixed with a recombinant tumor-associated antigen to induce immune responses and protective immunity in mice

The cationic liposome DOTAP is a well-known transfection reagent. It has been manufactured and approved for clinical use, is readily available, and can be easily used as an adjuvant. These characteristics prompted us to investigate the effectiveness of DOTAP as an adjuvant to induce immune responses and protective immunity in mice using baculovirus-derived carcinoembryonic antigen (bV-CEA) as a model antigen. Two routes of administration and a dose-response study of bV-CEA were used in BALB/c mice to define the magnitude of the immune response as well as the most effective route of immunization. The results demonstrate differences in antibody titers, immunoglobulin (Ig)G isotype, and T-cell responses between the intravenous (i.v.) or subcutaneous (s.c.) route of immunization. The titer of the anti-CEA antibodies induced by the s.c. immunization was greater than the response by i.v. immunization. The s.c. route enhanced the IgG2a/2b isotype, whereas i.v. immunization elicited primarily IgG1. T-cell proliferation responses and cytokine production paralleled the humoral response (i.e., production was higher in the s.c. immunized animals). No differences in immunological responses were seen using either 25 or 10 microg of bV-CEA three times. An amount of 25 microg of bV-CEA/DOTAP given by s.c. immunization was sufficient in protecting mice from the transplant of syngeneic tumor cells transduced with the human CEA gene. We conclude that the cationic liposome DOTAP may be a useful immunoadjuvant for active anti-tumor immunotherapy in future clinical trials. This study will help to define the most effective way to use such an adjuvant.     

The use of monoclonal antibody for the immunodiagnosis of Fasciola gigantica infection in cattle

Antigens that were specific to Fasciola gigantica were obtained from the whole worm homogenate of the parasite by immunoaffinity chromatography in cyanogen bromide-activated sepharose 4B columns and used for the production of monoclonal antibodies. The F. gigantica-specific monoclonal antibody was labelled with horseradish peroxidase and used for the detection of circulating antigen by the direct ELISA method in the sera of cattle experimentally infected with the parasite. Circulating antigens were detectable in the sera of the animals as from the third week after infection while negative absorbance values were obtained 2 weeks after the termination of the infection by chemotherapy with oxyclozanide. This immunodiagnostic method offers an attractive alternative as a supplement to the conventional coprological diagnosis of fasciolosis.     

Evaluation of electrophoretic immunoblotting for the detection of antibodies against the bovine leukosis virus in cattle

Six antigen preparations of bovine leukemia virus, including affinity-purified glycoprotein gp51, gradient-purified fetal lamb kidney-bovine leukemia virus antigen, and four crude antigens, were used in combination with several groups of cattle sera, for the evaluation of electrophoretic immunoblotting as a serological test method. Sera (89) from cattle naturally-infected with bovine leukosis virus, a panel of reference sera from infected and uninfected cattle (18), and serial bleedings from experimentally-infected cows (4) were used. Major differences between the six antigen preparations were observed in their reactivity with the various sera. The immunological variabilities of these antigens were confirmed further by their reactions with a gp51-specific monoclonal antibody. The known immunodominant gp51 failed as a reliable indicator for the serological status of the sera in blots when compared to the results on the same sera, two gp51-specific ELISAs and the agar gel immunodiffusion test were used as reference tests. There was a lack of staining of gp51 antigen by many sera, probably due to the labile nature of the gp51 molecule. On the other hand, non-specific staining in the gp51 region appeared with high frequency in some antigens. Antibody staining of the internal viral protein p24 correlated well with the results of the three reference tests. Other bands stained infrequently and were of no diagnostic value.     

Giardiasis in travellers: evaluation of an antigen-capture ELISA for the detection of Giardia lamblia-antigen in stool

Diagnosis of giardiasis relies largely on the microscopical detection of trophozoites or cysts in feces. However, this method is labour- and time-intensive and depends highly on the skill of an experienced microscopist. In order to identify the prevalence of Giardia lamblia in travellers returning from overseas, we evaluated sensitivity and specificity of a commercially available ELISA kit for the detection of Giardia-lamblia-antigen in stool. Nine hundred seventy-eight stool samples from 795 patients were examined by microscopy (iron-hematoxyilin-stain, SAF-concentration) and ELISA. Altogether, Giardia infection could be detected in 74 subjects. On evaluation of all samples, the ELISA turned out to be more sensitive than microscopy (95.5% vs. 81.8%) and 99.7% specific for Giardia lamblia. Especially with microscopy, the examination of more than one stool specimen did improve diagnostic sensitivity. It seems therefore advisable to retain the practise of examining at least three stool samples before considering a patient as not infected. The coproantigen-ELISA is especially advantageous in situations where only a single stool sample can be examined. It should not, however, replace microscopical examination of stool specimens for ova and parasites since other potential pathogens would otherwise escape detection.     

Location of induction and expression of protective immunity against Fasciola hepatica at the gut level: a study using an ex vivo infection model with ligated gut segments

PURPOSE: We developed a semiautomatic class solution to irradiate centrally located Stage III non-small cell lung cancer (NSCLC), involving a beam intensity modulation technique and optimization using a biophysical cost function. METHODS AND MATERIALS: Treatment for 10 patients with Stage III NSCLC was planned, using a conventional three- or four-beam three-dimensional (3D) technique and two techniques involving, respectively, seven (BIM1) and five (BIM2) noncoplanar beam incidences with intensity modulation. Two planning target volumes were defined: PTV1 included macroscopic tumor volume and PTV2 included macroscopic and microscopic disease. Beams were divided into beam parts (segments) and their outlines were defined during virtual simulation. Optimization using a biophysical cost function determined beam weights, segment weights, and wedge angles. Biological end points included tumor control probability of both target volumes (TCP1 and TCP2) and normal tissue complication probability (NTCP) of heart, lung, and spinal cord. The resulting uncomplicated local control probability (UCLP) was calculated. Physical end points included dose at PTV1 expressed as a dose minimum and dose maximum. Target-dose inhomogeneity was constrained in all plans. RESULTS: Concerning tumor evaluation, TCP1 was 74% (range 54-89%) for the 3D plan, 78.0% (range 62-94%) for BIM1, and 86.0% (range 59-93%) for BIM2. TCP1*TCP2 was, respectively, 67.0% (range 39-81%), 73.0% (range 56-94%), and 81.0% (range 54-93%). Minimum doses to PTV1 were 85, 80, and 88 Gy with the three respective techniques, while dose maxima were 89, 101, and 100 Gy. NTCPs of lung were 45.0% (range 11-75%) for 3D, 19.5% (range 8-59%) for BIM1, and 24.5% (range 3-61%) for BIM2. NTCPs of heart and spinal cord were comparable for all techniques. ULCPs were 37.0% (range 9-73%), 52.5% (range 22-86%), and 60.0% (range 20-85%), respectively. Applying physical limits to ensure clinical safety, minimum doses at PTV1 were recalculated. These were 72, 71, and 74 Gy for 3D, BIM1, and BIM2, respectively. CONCLUSION: The BIM2 plan is a candidate class solution for dose escalation studies in centrally located Stage III NSCLC.     

The use of SPECT with 201Tl in the evaluation of brain tumors

There is increasing interest in the literature regarding the use of SPECT (single photon emission computed tomography) with 201Tl in the evaluation of cerebral tumours. Thallium (201Tl) is one of the isotopes most commonly used in studies of the myocardium and in the diagnosis of various tumours. Normal brain takes up very little 201Tl, but in viable tumour tissue the 201Tl becomes intracellular and is rapidly cleared from the blood stream. Such cerebral tumour uptake is related to changes in the permeability of the blood-brain barrier, regional blood flow and transport by means of the Na(+)-K+ adenosine triphosphate pump. Once the SPECT images have been obtained following administration of the radioisotope, one can calculate the indices of early and of late uptake together with the retention index. The usefulness of SPECT in the preoperative detection of malignant expansive pathology, choice of tumour region during stereotaxic biopsy, control of tumour reproduction and their differentiation from post-radiotherapy necrotic tissue, and evaluation of chemotherapy in treatment of cerebral tumours are analyzed. Finally the application of SPECT in metastatic conditions and the differential diagnosis of expansive lesions is considered.     

Physiologic studies with the sulfate-reducing bacterium Desulfovibrio desulfuricans: evaluation for use in a biofuel cell

The growth kinetics of the sulfate-reducing bacteria Desulfovibrio desulfuricans Essex 6 was investigated under various conditions for potential use in a microbial fuel cell that recovers electrons generated from the reduction of sulfate to hydrogen sulfide. Hydrogen sulfide was found to inhibit growth and decrease both the growth yields and the sulfate-specific reduction rate. Hydrogen sulfide inhibition was direct, reversible, and not due to limitation by iron deficiency. A high initial lactate concentration also retarded bacterial growth, reduced the specific sulfate reduction rates, and gave variable biomass growth yields. This effect resulted from a bottleneck in the lactate oxidation pathway which induced the production of the secondary product butanol. The use of pyruvate as a carbon source was more advantageous than lactate in terms of growth rate and biomass growth yields, with only a slight decrease in the rate of specific sulfate reduction. For equal biomass, a slightly higher current density was generated from lactate than pyruvate, but pyruvate required nearly 40% less sulfate.     

A simple, quantitative, reproducible avidin-biotin ELISA for the evaluation of group B streptococcus type-specific antibodies in humans

Type-specific antibodies to the capsular polysaccharides (CP) of group B Streptococcus (GBS) are protective. Historically, the radioactive antigen-binding assay (RABA) has been used to determine GBS antibody levels. This method measures total immunoglobulin and employs the use of radioactive materials. We have developed an avidin-biotin ELISA that is less hazardous and is able to measure GBS Ia, Ib, II or III CP specific IgG. To avoid inconsistent binding to the plate, the CPs from GBS Ia, Ib, II and III were derivatized using adipic acid dihydrazide (ADH) and subsequently biotinylated without altering their antigenic epitopes and bound to avidin coated plates. Plasma from three different human subjects immunized with a tetravalent CP vaccine were used to prepare IgG references for Ia, II and III, respectively, thus rendering the assay quantitative for those types. The assay is able to detect nanograms per milliliter of GBS Ia, Ib, II or III specific antibody. This method is reproducible, sensitive and correlates with RABA by 76%.