Search

Abstract Background A fundamental question that arises during epidemiological investigations of bacterial disease outbreaks is whether the outbreak strain is genetically related to a proposed index strain. Highly discriminating genetic markers for characterizing bacterial strains can help in clarifying the genetic relationships among strains. Under the auspices of the European Society of Clinical Microbiology and Infectious Diseases, the European Study Group for Epidemiological Markers (ESGEM) established guidelines for evaluating the performance of typing systems based of a number of criteria. Recently, HOOF-Print genotype analysis, a new method for typing Brucella abortus strains based on hypervariability at eight tandem repeat loci, was described. This paper evaluates the HOOF-Print assay by four of the criteria set out by the ESGEM: typeability, reproducibility, power of discrimination, and concordance with other typing methods. Results The HOOF-Print Assay was evaluated with a test population composed of 97 unrelated field isolates and 6 common laboratory strains of B. abortus. Both typeability and reproducibility of the assay were excellent. Allele diversity and frequency varied widely among the eight loci, ranging from 1 to 13 alleles. The power of discrimination, measured by the Hunter-Gaston discrimination index (HGDI), varied by locus ranging from 0 to 0.89, where a maximal value of 1.0 indicates discrimination of all strains. The HGDI values calculated for subgroups sorted by biovar were similar to the values determined for the whole population. None of the individual loci achieved the recommended HGDI threshold of 0.95, but the HGDI of the composite profiles was 0.99 (93 unique genotypes from 97 field strains evaluated), well above the recommended threshold. By comparison, the HGDI value for biovar typing was 0.61 in a test population biased with disproportionate numbers of the less common biovars. Cluster analysis based on HOOF-Print genotypes assembled the strains into hierarchical groups with no apparent association with the time or location of strain isolation. Likewise, these hierarchical groups were not homogeneous with regard to biotype. In one extreme case, two field isolates with identical fingerprints were identified as different biovars by conventional methods. Conclusion The main purpose of this study was to assess the ability of HOOF-Print genotyping to discriminate unrelated field strains of B. abortus, and whether the assay met established requirements for bacterial strain typing methods. The discriminatory power of the assay was remarkable, considering the genetic homogeneity found among species within the genus. The assay met or exceeded all of the recommended levels for the performance criteria of typeability, reproducibility, and power of discrimination, however some inconsistencies with conventional biovar typing were observed. Nevertheless, the results indicate that with cautious interpretation, multilocus genotyping of polymorphic tandem repeats by HOOF-Print analysis could be a valuable complement to routine epidemiological investigations into localized B. abortus outbreaks.

Abstract Background Currently, there are very few tools available for subtyping Brucella isolates for epidemiological trace-back. Subtyping is difficult because of the genetic homogeneity within the genus. Sequencing of the genomes from three Brucella species has facilitated the search for DNA sequence variability. Recently, hypervariability among short tandem repeat sequences has been exploited for strain-typing of several bacterial pathogens. Results An eight-base pair tandem repeat sequence was discovered in nine genomic loci of the B. abortus genome. Eight loci were hypervariable among the three Brucella species. A PCR-based method was developed to identify the number of repeat units (alleles) at each locus, generating strain-specific fingerprints. None of the loci exhibited species- or biovar-specific alleles. Sometimes, a species or biovar contained a specific allele at one or more loci, but the allele also occurred in other species or biovars. The technique successfully differentiated the type strains for all Brucella species and biovars, among unrelated B. abortus biovar 1 field isolates in cattle, and among B. abortus strains isolated from bison and elk. Isolates from the same herd or from short-term in vitro passage exhibited little or no variability in fingerprint pattern. Sometimes, isolates from an animal would have multiple alleles at a locus, possibly from mixed infections in enzootic areas, residual disease from incomplete depopulation of an infected herd or molecular evolution within the strain. Therefore, a mixed population or a pool of colonies from each animal and/or tissue was tested. Conclusion This paper describes a new method for fingerprinting Brucella isolates based on multi-locus characterization of a variable number, eight-base pair, tandem repeat. We have named this technique "HOOF-Prints" for Hypervariable Octameric Oligonucleotide Finger-Prints. The technique is highly discriminatory among Brucella species, among previously characterized Brucella strains, and among unrelated field isolates that could not be differentiated by classical methods. The method is rapid and the results are reproducible. HOOF-Printing will be most useful as a follow-up test after identification by established methods since we did not find species-specific or biovar-specific alleles. Nonetheless, this technology provides a significant advancement in brucellosis epidemiology, and consequently, will help to eliminate this disease worldwide.

Triple negative breast cancer (TNBC) is a type of breast cancer associated with early metastasis, poor prognosis, high relapse rates, and mortality. Previously, we demonstrated that SYA013, a selective σ2RL, could inhibit cell proliferation, suppress migration, reduce invasion, and induce mitochondria-mediated apoptosis in MDA-MB-231 cell lines, although we were unable to demonstrate the direct involvement of sigma receptors. This study aimed to determine the anticancer properties and mechanisms of action of SYA014, [4-(4-(4-chlorophenyl)-1,4-diazepan-1-yl)-1-(4-fluorophenyl)butan-1-one oxime], an oxime analogue of SYA013, the contribution of its sigma-2 receptor (σ2R) binding, and its possible synergistic use with cisplatin to improve anticancer properties in two TNBC cell lines, MDA-MB-231 (Caucasian) and MDA-MB-468 (Black). In the present investigation, we have shown that SYA014 displays anticancer properties against cell proliferation, survival, metastasis and apoptosis in the two TNBC cell lines. Furthermore, a mechanistic investigation was conducted to identify the apoptotic pathway by which SYA014 induces cell death in MDA-MB-231 cells. Since SYA014 has a higher binding affinity for σ2R compared to σ1R, we tested the role of σ2R on the antiproliferative property of SYA014 with a σ2R blockade. We also attempted to evaluate the combination effect of SYA014 with cisplatin in TNBC cells.

Knowledge of longitudinal electron bunch profiles is vital to optimize the performance of plasma wakefield accelerators and x-ray free electron laser linacs. Because of their importance to these novel applications, noninvasive frequency domain techniques are often employed to reconstruct longitudinal bunch profiles from coherent synchrotron, transition, or undulator radiation measurements. In this paper, we detail several common reconstruction techniques involving the Kramers-Kronig phase relationship and Gerchberg-Saxton algorithm. Through statistical analysis, we draw general conclusions about the accuracy of these reconstruction techniques and the most suitable candidate for reconstructing well-isolated longitudinal bunch profiles from spectroscopic data.

We have previously reported that dual 5-HT 1A and 5-HT 7 receptor ligands might find utility as treatment options for various CNS related conditions including cognitive and anxiolytic impairments. We have also more recently reported that SYA16263 has antipsychotic-like properties with an absence of catalepsy in animal models ascribed to its ability to recruit β-arrestin to the D 2 receptor. However, SYA16263 also binds with very high affinity to 5-HT 1A R (Ki = 1.1 nM) and a moderate affinity at 5-HT 7 R (Ki = 90 nM). Thus, it was of interest to exploit its pharmacophore elements in designing new dual receptor ligands. Using SYA16263 as the lead molecule, we have conducted a limited structure-affinity relationship (SAFIR) study by modifying various structural elements in the arylalkyl moiety, resulting in the identification of a new dual 5-HT 1A R and 5-HT 7 R ligand, 6-chloro-2-methyl-2-(3-(4-(pyridin-2-yl)piperazin-1-yl)propyl)-2,3-dihydro-1H-inden-1-one (21), which unlike SYA16263, has a sub-nanomolar (5-HT 1A R, Ki = 0.74 nM) and a low nanomolar (5-HT 7 R, Ki = 8.4 nM) affinity for these receptors. Interestingly, 21 is a full agonist at 5-HT 1A R and antagonist at the 5-HT 7 R, functional characteristics which point to its potential as an antidepressant agent., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Masson SAS.)

The RB51 vaccine strain of Brucella abortus , which confers safe and effective protection of cattle from B. abortus infection, was originally generated via serial passage of B. abortus 2308 to generate spontaneous, attenuating mutations. While some of these mutations have been previously characterized, such as an insertional mutation in the wboA gene that contributes to the rough phenotype of the strain, a comprehensive annotation of genetic differences between RB51 and B. abortus 2308 genomes has not yet been published. Here, the whole genome sequence of the RB51 vaccine strain is compared against two available 2308 parent sequences, with all observed single nucleotide polymorphisms, insertions, and deletions presented. Mutations of interest for future characterization in vaccine development, such as mutations in eipA and narJ genes in RB51, were identified. Additionally, protein homology modeling was utilized to provide in silico support for the hypothesis that the RB51 capD mutation is the second contributing mutation to the rough phenotype of RB51, likely explaining the inability of wboA -complemented strains of RB51 to revert to a smooth phenotype., (Copyright © 2020 Bricker et al.)

Our previous study has revealed 4-(4-(4-chlorophenyl)-1,4-diazepan-1-yl)-1-(4-fluorophenyl)butan-1-one·2HCl (SYA013) 1 as a sigma ligand with moderate selectivity for the sigma-2 receptor. Given the overexpression of sigma receptors in solid tumors and reports of sigma ligands with anticancer activities, we selected 1 for evaluation in several solid tumor cell lines. In addition, we have synthesized new analogs of 1 and now report that several of them bind preferentially at the sigma-2 receptor and have shown inhibition of several cancer cell lines including MDA-MB-231, MDA-MB-486, A549, PC-3, MIA PaCa-2 and Panc-1 cells. In particular, compounds 1 and 12 have demonstrated sub-micromolar activity against the Panc-1 cell line. It has also been observed that several of these compounds demonstrate selective toxicity toward cancer cells, when compared to normal cells., (Published by Elsevier Ltd.)

Previous work from our labs has indicated that a tropane analog of haloperidol with potent D2 binding but designed to avoid the formation of MPP(+)-like metabolites, such as 4-(4-chlorophenyl)-1-(4-(4-fluorophenyl)-4-oxobutyl)pyridin-1-ium (BCPP(+)) still produced catalepsy, suggesting a strong role for the D2 receptor in the production of catalepsy in rats, and hence EPS in humans. This study tested the hypothesis that further modifications of the tropane analog to produce compounds with less potent binding to the D2 receptor than haloperidol, would produce less catalepsy. These tests have now revealed that while haloperidol produced maximum catalepsy, these compounds produced moderate to low levels of catalepsy. Compound 9, with the least binding affinity to the D2R, produced the least catalepsy and highest Minimum Adverse Effective Dose (MAED) of the analogs tested regardless of their affinities at other receptors including the 5-HT1AR. These observations support the hypothesis that moderation of the D2 binding of the tropane analogs could reduce catalepsy potential in rats and consequently EPS in man., (Published by Elsevier Ltd.)

The dopamine D4 receptor has been shown to play key roles in certain CNS pathologies including addiction to cigarette smoking. Thus, selective D4 ligands may be useful in treating some of these conditions. Previous studies in our laboratory have indicated that the piperazine analog of haloperidol exhibits selective and increased affinity to the DAD4 receptor subtype, in comparison to its piperidine analog. This led to further exploration of the piperazine moiety to identify new agents that are selective at the D4 receptor. Compound 27 (KiD4=0.84 nM) was the most potent of the compounds tested. However, it only had moderate selectivity for the D4 receptor. Compound 28 (KiD4=3.9 nM) while not as potent, was more discriminatory for the D4 receptor subtype. In fact, compound 28 has little or no binding affinity to any of the other four DA receptor subtypes. In addition, of the 23 CNS receptors evaluated, only two, 5HT1AR and 5HT2BR, have binding affinity constants better than 100 nM (Ki <100 nM). Compound 28 is a potentially useful D4-selective ligand for probing disease treatments involving the D4 receptor, such as assisting smoking cessation, reversing cognitive deficits in schizophrenia and treating erectile dysfunction. Thus, further optimization, functional characterization and evaluation in animal models may be warranted., (Copyright © 2014. Published by Elsevier Ltd.)

Haloperidol induced catalepsy was determined using the classic bar test and a new MED Associates Catalepsy Test Chamber instrument. The dose that produced an adverse effect in 50% of rats (AED50) for haloperidol was calculated using the instrument data as 0.29 mg/kg. Hand scoring of the video recordings gave AED50 values of 0.30 and 0.31 mg/kg, both well within the 95% CL of the instrument data. Clozapine was also evaluated and catalepsy was not detected up to 40 mg/kg. No significant difference was found between the instrument and hand scoring data. The instrument was useful for testing haloperidol and clozapine, relieving much of the tedium and variability experienced without its use. It was especially valuable at measuring shorter time periods, where the researcher cannot react as quickly. Finally, olanzapine was also evaluated. However, clenched forepaws and hind paws prevented the use of the instrument alone at higher doses. A backup stopwatch was used for the bar test in these cases. Some of the advantages and limitations are discussed. Results are also compared to the crossed-legs position (CLP) test for all three antipsychotics. While haloperidol gave similar results at all concentrations tested, clozapine deviated significantly at the highest dose (40 mg/kg) displaying catalepsy in the CLP test but not in the bar test. Olanzapine displayed catalepsy in rats significantly different from vehicle at 40 mg/kg in both the bar and CLP tests. However, the CLP test may be more suited to compounds with gripping problems which prevent the consistent grasping of the bar. Overall, the instrument was found to be a useful aid in conducting the bar test for catalepsy. The CLP test was found to complement the bar test under certain conditions and could provide additional data that might be missed by the bar test for compounds producing grasping problems., (Published by Elsevier Inc.)

SYA013, a homopiperazine analog of haloperidol, was further evaluated for antipsychotic potential using additional animal models. Previously, SYA013 was tested in mice with an antipsychotic screening model in which it inhibited apomorphine induced climbing behavior, indicating antagonism of the dopaminergic system and the potential for use in the treatment of schizophrenia. In this study, SYA013 was shown to inhibit both d-amphetamine-induced locomotor activity in rats and conditioned avoidance response (CAR) in rats in a dose dependent manner and in the case of CAR, without producing any escape failure responses (EFRs), two tests predictive of antipsychotic action. The selective 5HT(1A) antagonist WAY100,635 was used to determine if binding of SYA013 to the 5HT(1A) receptor contributed to suppression of CAR. The results indicated that 0.63mg/kg WAY100,635 did not have a significant effect on the inhibition of CAR by SYA013. Pharmacokinetic parameters in brain and plasma were determined for SYA013. A log brain/plasma concentration ratio at a t(max) of 1.48 suggests that SYA013 readily crosses the blood brain barrier (BBB). The hypothesis that binding of SYA013 to the 5HT(1A) receptor contributed to the lack of significant catalepsy was investigated using the 5HT(1A) antagonist WAY100,635. The results of acute and semi-chronic tests suggest that binding to the 5HT(1A) receptor alone did not significantly account for the lack of catalepsy. Lack of catalepsy was preserved after the semi-chronic challenge with SYA013. These tests further indicate that SYA013 has a pharmacological profile with the potential for use in the treatment of neuropsychiatric diseases. In addition, the 5HT(1A) receptor does not appear to play a significant role in the pharmacological profile of SYA013., (Published by Elsevier Inc.)

Identifying the source of infectious disease outbreaks is difficult, especially for pathogens that infect multiple wildlife species. Brucella spp. are among the most problematic zoonotic agents worldwide, and they are notoriously difficult to detect and identify. We genotyped 10 variable number of tandem repeat (VNTR) DNA loci in 56 Brucella abortus isolates from bison (Bos bison), elk (Cervus elaphus), and cattle (Bos taurus) to test the wildlife species most likely to be the origin of recent outbreaks of brucellosis in cattle in the Greater Yellowstone Area. Isolates from cattle and elk were nearly identical but highly divergent from bison isolates. These data suggest elk, not bison, are the reservoir species of origin for these cattle infections. This study illustrates the potential power of VNTR genotyping to assess the origin of disease outbreaks, which are increasing worldwide following habitat fragmentation, climate change, and expansion of human and livestock populations.

The synthesis and exploration of novel butyrophenones have led to the identification of a diazepane analogue of haloperidol, 4-[4-(4-chlorophenyl)-1,4-diazepan-1-yl]-1-(4-fluorophenyl)butan-1-one (compound 13) with an interesting multireceptor binding profile. Compound 13 was evaluated for its binding affinities at DA subtype receptors, 5HT subtype receptors, H-1, M-1 receptors and at NET, DAT, and SERT transporters. At each of these receptors, compound 13 was equipotent or better than several of the standards currently in use. In in vivo mouse and rat models to evaluate its efficacy and propensity to elicit catalepsy and hence EPS in humans, compound 13 showed similar efficacy as clozapine and did not produce catalepsy at five times its ED(50) value.

The Brucella abortus strain S19, a spontaneously attenuated strain, has been used as a vaccine strain in vaccination of cattle against brucellosis for six decades. Despite many studies, the physiological and molecular mechanisms causing the attenuation are not known. We have applied pyrosequencing technology together with conventional sequencing to rapidly and comprehensively determine the complete genome sequence of the attenuated Brucella abortus vaccine strain S19. The main goal of this study is to identify candidate virulence genes by systematic comparative analysis of the attenuated strain with the published genome sequences of two virulent and closely related strains of B. abortus, 9-941 and 2308. The two S19 chromosomes are 2,122,487 and 1,161,449 bp in length. A total of 3062 genes were identified and annotated. Pairwise and reciprocal genome comparisons resulted in a total of 263 genes that were non-identical between the S19 genome and any of the two virulent strains. Amongst these, 45 genes were consistently different between the attenuated strain and the two virulent strains but were identical amongst the virulent strains, which included only two of the 236 genes that have been implicated as virulence factors in literature. The functional analyses of the differences have revealed a total of 24 genes that may be associated with the loss of virulence in S19. Of particular relevance are four genes with more than 60 bp consistent difference in S19 compared to both the virulent strains, which, in the virulent strains, encode an outer membrane protein and three proteins involved in erythritol uptake or metabolism.

Enantiomeric separation of the racemic 4-{3-(4-chlorophenyl)-3-hydroxypyrrolidin-1-yl}-1-(4-fluorophenyl)butan-1-one, a pyrrolidine analog of haloperidol, {(+/-)-SYA 09}, and subsequent binding studies revealed that most of the binding affinity at dopamine and serotonin receptors resides in the (+)-isomer {(+)-SYA 09} or the eutomer. Further pharmacological evaluation of the eutomer revealed that it has a higher affinity for the dopamine D4 (DAD4) receptor subtype (Ki = 3.6 nM) than for the DAD2 subtype (Ki = 51.1 nM) with a ratio of 14.2 (D2Ki/D4Ki ratio = 14.2). In an animal model of antipsychotic efficacy, the (+)-SYA 09 was efficacious with an ED50 value of 1.6 mg/kg, i.p., and at twice this value, (+)-SYA 09 did not induce significant catalepsy in rats.

The Brucella AMOS PCR assay was previously developed to identify and differentiate specific Brucella species. In this study, an abbreviated Brucella AMOS PCR test was evaluated to determine its accuracy in differentiating Brucella abortus into three categories: field strains, vaccine strain 19 (S19), and vaccine strain RB51/parent strain 2308 (S2308). Two hundred thirty-one isolates were identified and tested by the conventional biochemical tests and Brucella AMOS PCR. This included 120 isolates identified as B. abortus S19, 9 identified as B. abortus strain RB51, 57 identified as B. abortus biovar 1, 15 identified as B. abortus bv. 2, 1 identified as B. abortus bv. 2 (M antigen dominant), 7 identified as B. abortus bv. 4, and 22 identified as B. abortus S2308 and isolated from experimentally infected cattle. The Brucella AMOS PCR correctly identified each isolate as RB51/S2308, S19, or a field strain of Brucella.

A multiplex amplification and detection platform for the diagnosis of Mycobacterium bovis and Brucella abortus infection simultaneously in bovine milk and nasal secretions was developed. This system (designated the bovine pathogen detection assay [BPDA]-PCR) consists of duplex amplification of species-specific targets (a region of the BCSP31K gene of B. abortus and a repeat-sequence region in the hsp65 gene of M. bovis, respectively). This is followed by a solid-phase probe capture hybridization of amplicons for detection. On the basis of spiking experiments with normal milk, the analytical sensitivity of the assay was 800 CFU equivalents/ml of milk for B. abortus and as low as 4 CFU equivalents per ml of milk for M. bovis. BPDA-PCR was validated with 45 liver samples from lemmings experimentally infected with B. abortus. The assay sensitivity, based on culture status as a "gold standard," was 93.9%. In this experiment, BPDA-PCR also identified five culture-negative liver samples as positive (41.7%). Field studies for the evaluation of BPDA-PCR were performed with samples from dairy animals from geographically distinct regions (India, Mexico, and Argentina). A high prevalence of shedding of B. abortus (samples from India) and M. bovis (samples from Mexico) was identified by BPDA-PCR. In samples from India, B. abortus shedding was identified in 86% of milk ring test-positive animals (n = 15) and 80% of milk ring test-negative cows (n = 5). In samples from Mexico, M. bovis was identified by PCR in 32.6% of pools (n = 46) of milk that each contained milk from 10 animals and in 56.2% of nasal swabs (n = 121) from cattle from tuberculin test-positive herds. In contrast, the Argentine cattle (n = 70) had a modest prevalence of M. bovis shedding in nasal swabs (2.9%) and milk (1.4%) and of B. abortus in milk (11.4%). On the basis of these analyses, we identify BPDA-PCR as an optimal tool for both screening of herds and testing of individual animals in a disease eradication program. A combination of the duplex assay, screening of milk samples in pools, and the proposed algorithm provides a highly sensitive, cost-effective, and economically viable alternative to serological testing.

Recently, gram-negative bacteria isolated from a variety of marine mammals have been identified as Brucella species by conventional phenotypic analysis. This study found the 16S rRNA gene from one representative isolate was identical to the homologous sequences of Brucella abortus, B. melitensis, B. canis, and B. suis. IS711-based DNA fingerprinting of 23 isolates from marine mammals showed all the isolates differed from the classical Brucella species. In general, fingerprint patterns grouped by host species. The data suggest that the marine mammal isolates are distinct types of Brucella and not one of the classical species or biovars invading new host species. In keeping with historical precedent, the designation of several new Brucella species may be appropriate.

Many bacteria are difficult to subtype due to high genetic relatedness. In the cases of pathogens of medical or veterinary importance, subtyping is an essential tool of epidemiologists. This report describes a method for molecular subtyping based on the detection of point mutations without DNA sequencing or specialized equipment. The method, known as RNA mismatch cleavage, hybridizes RNA transcripts derived from PCR-amplified DNA, with a control RNA transcript followed by RNase cleavage at point-mutation mismatches. The method was successful in distinguishing all six Brucella species tested and was able to distinguish 11 of the 18 biovars studied. Of the remaining seven biovars (all of which are Brucella abortus strains), three subgroups were identified. The method should be applicable to all hard-to-subtype bacterial strains.

High performance liquid chromatography (HPLC) has proven to be an extremely useful analytical technique to separate and identify different types of hemoglobins, particularly A, C, F and S in blood samples, and compute their relative percentages. Such data provide useful information in the diagnosis of hemoglobinopathies including sickle cell anemia, beta-thalassemia, hemoglobin C disease,etc. In the present investigation, we have explored the determination of absolute concentrations of individual hemoglobins in g/dL and recommend it as an additional parameter which could be included as part of clinical data. Possible correlations between g/dL hemoglobin and the severity of a specific hemoglobinopathy could be established and aid in the diagnosis and/or treatment of such diseases. Several commercially available hemoglobin standards were analyzed and evaluated for use in creating g/dL calibration graphs for the HPLC. Appropriate calibration procedures have been developed and are presented. Also, linear regression graphs and sample chromatograms are included. Preliminary results obtained demonstrate the feasibility of using commercially available hemoglobin standards to calibrate an HPLC method for the estimation of absolute hemoglobin concentrations.

Two repeated DNA elements of 103 bp and 105 bp were discovered in brucellae and designated Bru-RS1 and Bru-RS2, respectively. The two elements are palindromic, are 65% similar in sequence, form two families of elements that are slightly divergent in sequence, appear to be intergenic, and are found, collectively, in more than 35 copies in brucellae. These elements are bounded by perfect or nearly perfect inverted repeats. A third copy of the terminal repeat is found within the elements and is the terminus for several truncated copies of the Bru-RS1 family. Hybridization patterns for the elements among brucellae were unique. The elements are dispersed, highly conserved among brucellae, and hot-spots for insertion by IS711.

Field-caught adult male and femaleAedes hendersoni are difficult to distinguish from the sibling speciesA. triseriatus. We found that mosquitoes from the same sex of the sibling species can not be readily separated either by unique cuticular hydrocarbon components or by differences in percent composition of those components. Multivariate analysis of the cuticular hydrocarbon data does not provide good separation. Cuticular hydrocarbons were identified using gas chromatography electron-impact mass spectrometry and gas chromatography chemical-ionization mass spectrometry. Flame-ionization capillary gas chromatography was used for quantitative analysis of individual mosquitoes. Sixty-four hydrocarbons with chain lengths from C16 to greater than C46 were common to both species. Identified hydrocarbon components weren-alkanes, monomethylalkanes, dimethylalkanes, trimethylalkanes, and alkenes.

A Brucella abortus protein with a molecular weight of 50 kDa has been shown to bind bovine immunoglobulin G from healthy, brucellosis-free animals. The Brucella immunoglobulin G binding molecule appears to be a protein, since it is susceptible to proteolysis. The protein is presumed to be located on the cell surface, since intact cells precipitate bovine immunoglobulin G. Examination of other species of Brucella shows that all Brucella species and strains tested express the protein. B. abortus cells also bound immunoglobulin G from other animal species. These included cat, chicken, dog, guinea pig, horse, human, mouse, rat, sheep, swine, and turkey but not immunoglobulin G from goat or rabbit.

A study was conducted to determine whether subcomponent proteins (previously identified as BCSP20, BCSP31, and BCSP45, and the corresponding recombinant proteins rBCSP20, rBCSP31, and rBCSP45) that were recovered from the cell surface of Brucella abortus strain 19 were immunogenic and protective for mice when compared with Brucella cell surface protein (BCSP) and with a proteinase K-treated lipopolysaccharide (PKLPS) extracted from B abortus strain 2308. Protection was evaluated after challenge exposure with a virulent culture of B abortus strain 2308, using CD-1 or BALB/c mice or both inoculated with vaccines of various combinations and concentrations, with and without PKLPS or BCSP. Protection was assessed by enumeration of splenic colony-forming units, reduced mean splenic weight relative to controls, and the relative serologic responses (immune response) in an ELISA. The general results indicate that BCSP, PKLPS, BCSP20, and BCSP31 are immunogenic or protective or both. Protectiveness was not observed for each of the recombinant proteins; however, results from the combined recombinant protein vaccine study suggest the immunogenicity of the recombinant proteins. The apparent immune-inducing properties of BCSP20 and BCSP31 are thought to be attributable to the presence of an immunogenic and protective BCSP fraction (possibly lipopolysaccharide) still associated. Serologic results support our conclusion that each of the recombinant protein vaccines did not induce a protective response comparable to that of BCSP or PKLPS, even when the subcomponents were combined. Although the results suggest that the subcomponents of BCSP apparently induced partial protection, they are thought to be only a part of the antigens contained in BCSP that influence the serologic response.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunogenic or pathogenic factors of recombinant proteins (rBCSP20, rBCSP-31, and rBCSP45 of Brucella abortus strain 19) for mice were compared with factors of a proteinase K-treated lipopolysaccharide extracted from B abortus strain 2308. Mice were vaccinated with 4 products, using different inoculation schedules and were challenge exposed with a virulent culture of B abortus strain 2308. Blood samples were collected 2 weeks after vaccination and at necropsy and sera were obtained. Spleens were cultured for B abortus at necropsy (3 to 4 weeks after challenge exposure). Mice given proteinase K-treated lipopolysaccharide alone or in conjunction with rBCSP20 or rBCSP45 proteins were protected, but mice given rBCSP31 on the same day as challenge exposure were not. Vaccination with recombinant proteins alone neither provide protection nor significantly (P greater than 0.05) increase the pathogenic effect of the challenge-exposure culture. Seemingly, rBCSP31 might be a virulence factor of B abortus.

Of the nine antigenic determinants on the glycoprotein (G) of the New Jersey serotype of vesicular stomatitis virus (VSV) identified by competitive binding of 25 monoclonal antibodies (MAbs), those relegated to epitopes I, II, III, and IV exhibited no significant ability to neutralize virus infectivity but some nonneutralizing MAbs cross-reacted by ELISA with the G protein of VSV-Indiana. High-titered neutralization of homotypic virus was exhibited by epitope V, VI, and VII MAbs but quite variable neutralizing activity was found among MAbs of epitope "family" VIII and particularly the heterogeneous epitope "family" IX. Peptide mapping of the epitopes was not feasible because most MAbs would not bind by Western blotting to G protein under standard conditions of proteolysis or disulfide bond reduction. Therefore, a technique was devised for roughly locating epitopes by protease footprinting of G protein partially protected by individual MAbs complexed with staphylococcal protein A-Sepharose beads. Under these conditions, MAbs to all nine epitopes protected a similar 12-kDa fragment of the G protein from proteolysis by Staphylococcus aureus V8 protease. N-Terminal amino acid sequencing mapped two of these 12-kDa peptide footprints to a position on the G protein extending from amino acid 219 to about 100 amino acids downstream. Although MAbs to only one epitope bound to the 12-kDa fragment by Western blotting, these data suggest, but do not prove, that all nine epitopes of the undenatured VSV-New Jersey G protein are clustered at the middle 20% of a highly structured protein. This method may help to identify the general regions for epitopes on complex proteins of as yet unknown three-dimensional structure.

Brucella abortus is the causative agent for brucellosis in cattle and man. Development of a single diagnostic test for the differentiation of vaccinated from infected animals and the development of a nonviable 'subunit' vaccine are top priorities of the brucellosis research program in the United States. Preliminary evidence previously showed that a purified 31-kDa protein (thought to be localized at or near the bacterial cell surface) protects against experimental brucellosis in rodents. The gene for this 31-kDa protein has now been cloned in Escherichia coli. The protein is expressed well, apparently from its native promoter, when placed in several different E. coli plasmids. The nucleotide sequence of the flanking and encoding sequences has been determined, and comparison with the N-terminal amino acid (aa) sequence of the mature protein indicates the presence of a putative 28-aa signal sequence. The availability of the 31-kDa protein free of Brucella contaminants now allows rigorous study of the immunological properties of this protein.

A 31-kilodalton (kDa) protein extracted from Brucella abortus was previously cloned into Escherichia coli and expressed at high levels. The E. coli-derived protein can be purified by a simple 2-step procedure entailing detergent extraction followed by ion-exchange chromatography. Subsequent analyses show that the E. coli-derived protein is identical to the Brucella-derived protein in molecular weight and isoelectric point. A partial amino acid sequence of the N-terminus of the protein of E. coli origin matches the predicted sequence, based on DNA sequence data. Using specific antiserum raised against the E. coli-derived protein, 34 strains of Brucella, representing all 6 recognized species, were examined for expression of the 31-kDa protein by Western blotting. This protein was detectable in all, but one Brucella species (B. ovis), including all 8 biovars of B. abortus tested. This degree of conservation supports further study of the 31-kDa protein for potential exploitation as a vaccine or diagnostic component.