The Significance of NT1, NT2, and NT3 Antigens in Epidemiological Investigations of Bovine Group-B Streptococcus Infections

On typing of 90 strains of bovine Group-B streptococci (B-str.) from 21 herds, NT1, NT2 and NT3 antigens were found in 30 %, viz., NT1 in 14.4 %, NT2 in 3.3 %, and NT3 in 12.2 %. In 9 of the 21 herds examined (43 %) one or other of these antigens was found. Among 8 strains from 4 herds of Herd type NT the NT1 antigen was demonstrated once. In 5 herds of Herd type X, 20 strains were typed, and 9 isolates (45 %) from 3 herds (60 %) carried one or other of the three antigens. Among herds in which the herd type was referable to polysaccharide antigens of the accepted set, NT3 antigen was present in 1 herd of Herd type Ia, namely along with IaX antigens in two strains, and with Ia antigen in one. In 3 herds of Herd type Ibc and in 2 herds of Herd type Ib no NT1, NT2, or NT3 antigen was demonstrated. On the other hand, such antigens were found in all of 4 herds of Herd type III, being present in 50 % of the strains (14/28) examined, and the frequency of NT1, NT2, and NT3 antigens was only slightly higher among NT and X strains (10/19 or 53 %) than among III and IIIX strains (4/9 or 44 %). The NT2 reference strain carried the Ibc protein antigen. In addition to strong homologous reactions, NT1 antiserum reacted with B-str. group antigen, NT2 antiserum with Ibc antigen, and NT3 antiserum with the NT1 reference strain. It is concluded that the NT1, NT2, and NT3 antigens are of doubtful value in epidemiological studies on bovine infections with group-B streptococci.     

H-Y antigen: No evidence for alleles in wild strains of mice

H-Y antigen(s) coded or controlled by the Y chromosome in a variety of wild mouse strains have been compared with those of the inbred laboratory strains C57BL/6 (B6) and C57BL/10 (B10). H-Y antigen(s) were detected by H-2-restricted cytotoxic T cells from B6 and B10 female mice primed in vivo and boosted in vitro with syngeneic male spleen cells: There was no difference in the degree of H-Y specific lysis of male cells from the C57BL strains and of F₁ hybrids or B6 congenic mice carrying the Y chromosome from the wild mouse strains examined. This result indicated that at the level of target cell specificity the H-Y antigen(s) from wild and laboratory strains were indistinguishable. H-Y antigen(s) were also found to be indistinguishable at the level of the in vitro induction of the anti H-Y cytotoxic response: F₁ female mice, primed in vivo and boosted in vitro with homologous F₁ male cells, all made H-Y-specific responses and where it could be examined, the target cell specificity of the anti-H-Y cytotoxic cells showed that B10 male cells as well as the homologous F₁ male cells (where the Y chromosome was derived from the wild strain) were good targets. Finally, possible differences in H-Y transplantation antigens between the wild strains and the B10 laboratory strain were examined by grafting F₁ male mice, the progeny of B10 females, and wild strain males with B10 male skin. These grafts were not rejected during an observation period of more than 9 months. Taken together, neither the cytotoxic data nor the skin graft data provide any evidence for allelism of H-Y even though the mouse strains examined were collected from widely disparate geographical locations.     

Prevalence of Colonization Factor Antigens (CFAs) and Adherence to HeLa Cells in Enterotoxigenic Escherichia coli Isolated from Feces of Children in São Paulo

Fifty-eight enterotoxigenic Escherichia coli (ETEC) strains, isolated from children with and without diarrhea in Sao Paulo, were examined for the presence of colonization factor antigens (CFAs) and their ability to adhere to HeLa cells. Antisera to CFA/I, the coli surface (CS) antigens CS1CS3, CS2CS3, and CS2 of CFA/II, CFA/III, and CS5CS6 and CS6 of CFA/IV were used. CFAs were identified in 43% of the ETEC strains: 40% of the strains with CFAs harbored CFA/I, 24% carried CFA/II (CS1CS3), 24% carried CFA/IV (CS6), and 12% carried CFA/IV (CS5CS6). CFAs occurred mainly among ETEC strains producing only heat-stable (ST-I) enterotoxin and in strains also producing heat-labile toxin (LT-I). No ETEC strains tested expressed CFA/III. A marked change in serotypes of ST-I-producing strains was found in Sao Paulo between 1979 and 1990. Adherence to HeLa cells was detected in 14% of the ETEC strains. All of them had a diffuse adherence pattern and produced only ST-I, and 88% carried CS6 antigen.     

The type antigen III of group F streptococci separation of group and type antigens and partial characterization of type III antigen

Polysaccharides extracted from Z₃III streptococci either with formamide or with dilute hydrochloric acid or isolated from the growth medium could be fractionated in type III- and group Z₃ antigens by alcohol precipitation. No good separation could be obtained from TCA extracts. When the same extractions and fractionations were applied to streptococci carrying type III antigen, but different group antigens, good separations were again obtained of all formamide extracts, but not of all hydrochloric acid extracts. The group antigens showed a rhamnose content of at least 50% and contained hexosamines. Type III antigens contained mainly rhamnose, glucose and galactose in relative amounts of approximately 1:2:3. Analysis of the methylated type III antigen suggests it to be a polysaccharide with a linear structure. Type III antigen isolated from the medium was characterized not only by a different sugar ratio, but also by its fucose content of 20%. In some cases the purified polysaccharides contained considerable amounts of glycogen-like material. Partial acid hydrolysis of the type III antigen extracted with formamide yielded a great number of oligosaccharides. Analyses, inhibition reactions and methylation studies gave indications that the most probable structure of a determinant group of type III antigen is β-glucosyl-(1-6)-galactosyl-(1-6)-galactosyl-(1-3)-rhamnose. The possibility of the existence of a second determinant group is discussed.     

An enzyme-linked immunosorbent assay to detect alkali-soluble spore surface antigens of strains of Nosema bombycis (Microspora: Nosematidae)

Spore surface antigens of strains of Nosema bombycis were extracted with alkaline solutions and used in an indirect enzyme-linked immunosorbent assay. Treatment of N. bombycis spores with 0.1 n potassium carbonate or potassium hydroxide solution at 27°C for 30 min was sufficient for the extraction of the antigens. Usually, 10⁸ spores of N. bombycis liberated ca. 30 μg spore surface proteins. The indirect enzyme-linked immunosorbent assay detected as little as 60 ng of spore surface proteins (ca. 2000 spore-equivalent antigen). The alkali-soluble spore surface antigens of N. bombycis contained a specific antigen and were stable under storage at −20°C for more than 1 year. The serological assay separated the Nosema isolates pathogenic to the silkworm into three groups.     

Plasmid deoxyribonucleic acid in strains of Bacillus sphaericus and in Bacillus moritai

Bacillus moritai and six strains of Bacillus sphaericus pathogenic to dipteran larvae were examined for the presence of covalently closed circular (CCC) DNA. The plasmid profiles of the bacteria were analyzed using a cleared lysate electrophoresis technique. Four of the six strains of B. sphaericus examined contained CCC DNA. Strain SSII-1 contained two plasmids (pKA1, pKA2) having molecular weights of about 8.4 and 2.0 megadaltons (MDa). Strains 1404 and 1881 each contained one plasmid, pKA3 and pKA4, respectively. pKA3 had a molecular weight of about 8.2 MDa. pKA4 had a relatively large plasmid with a molecular weight of about 33.5 MDa. Strain K contained five size classes of CCC DNA. The plasmids pKA5, pKA6, pKA7, pKA8, and pKA9 had molecular weights of about 11.4, 10.9, 7.4, 7.0, and 6.4 MDa, respectively. Strains 1593-4 and 1691 were plasmidless and could not be distinguished from each other based on their plasmid profiles. B. moritai ATCC 21042 contained two size classes of CCC duplex DNA; pRF100 had a molecular weight of about 4.6 MDa and pRF101 had a molecular weight of about 2.1 MDa. No phenotype association with any of the isolated plasmids has been determined.     

Bordetella pertussis serotypes in Canada.

A study was done to determine the major antigenic factors of Bordetella pertussis strains isolated throughout Canada and whether these isolates have the same antigenic structure as the bacilli in the currently used vaccines. Testing for the major pertussis antigens, factors 1, 2 and 3, was conducted with 440 freshly isolated strains of B. pertussis received from seven canadian provinces between August 1976 and February 1978 and six batches of pertussis vaccine or immunizing agents containing pertussis vaccine. With the aid of specific antisera prepared in rabbits, the antigenic factors were detected by a slide agglutination technique. Almost all (98.9%) of the pertussis strains examined were serotype 1,3.All six batches of pertussis vaccine or immunizing agents containing pertussis vaccine proved to be rich in each of the three main pertussis agglutinogens.     

Plasmodium falciparum antigens on the surface of the gametocyte-infected erythrocyte

Background

The asexual blood stages of the human malaria parasite Plasmodium falciparum produce highly immunogenic polymorphic antigens that are expressed on the surface of the host cell. In contrast, few studies have examined the surface of the gametocyte-infected erythrocyte.

Methodology/Principal Findings

We used flow cytometry to detect antibodies recognising the surface of live cultured erythrocytes infected with gametocytes of P. falciparum strain 3D7 in the plasma of 200 Gambian children. The majority of children had been identified as carrying gametocytes after treatment for malaria, and each donated blood for mosquito-feeding experiments. None of the plasma recognised the surface of erythrocytes infected with developmental stages of gametocytes (I–IV), but 66 of 194 (34.0%) plasma contained IgG that recognised the surface of erythrocytes infected with mature (stage V) gametocytes. Thirty-four (17.0%) of 200 plasma tested recognised erythrocytes infected with trophozoites and schizonts, but there was no association with recognition of the surface of gametocyte-infected erythrocytes (odds ratio 1.08, 95% C.I. 0.434–2.57; P = 0.851). Plasma antibodies with the ability to recognise gametocyte surface antigens (GSA) were associated with the presence of antibodies that recognise the gamete antigen Pfs 230, but not Pfs48/45. Antibodies recognising GSA were associated with donors having lower gametocyte densities 4 weeks after antimalarial treatment.

Conclusions/Significance

We provide evidence that GSA are distinct from antigens detected on the surface of asexual 3D7 parasites. Our findings suggest a novel strategy for the development of transmission-blocking vaccines.     

Evaluations for In Vitro Correlates of Immunogenicity of Inactivated Influenza A H5, H7 and H9 Vaccines in Humans

Background

Serum antibody responses in humans to inactivated influenza A (H5N1), (H9N2) and A (H7) vaccines have been varied but frequently low, particularly for subunit vaccines without adjuvant despite hemagglutinin (HA) concentrations expected to induce good responses.

Design

To help understand the low responses to subunit vaccines, we evaluated influenza A (H5N1), (H9N2), (H7N7) vaccines and 2009 pandemic (H1N1) vaccines for antigen uptake, processing and presentation by dendritic cells to T cells, conformation of vaccine HA in antibody binding assays and gel analyses, HA titers with different red blood cells, and vaccine morphology in electron micrographs (EM).

Results

Antigen uptake, processing and presentation of H5, H7, H9 and H1 vaccine preparations evaluated in humans appeared normal. No differences were detected in antibody interactions with vaccine and matched virus; although H7 trimer was not detected in western blots, no abnormalities in the conformation of the HA antigens were identified. The lowest HA titers for the vaccines were <1∶4 for the H7 vaccine and 1∶661 for an H9 vaccine; these vaccines induced the fewest antibody responses. A (H1N1) vaccines were the most immunogenic in humans; intact virus and virus pieces were prominent in EM. A good immunogenic A (H9N2) vaccine contained primarily particles of viral membrane with external HA and NA. A (H5N1) vaccines intermediate in immunogenicity were mostly indistinct structural units with stellates; the least immunogenic A (H7N7) vaccine contained mostly small 5 to 20 nm structures.

Summary

Antigen uptake, processing and presentation to human T cells and conformation of the HA appeared normal for each inactivated influenza A vaccine. Low HA titer was associated with low immunogenicity and presence of particles or split virus pieces was associated with higher immunogenicity.     

Natural variation in immune responses to neonatal Mycobacterium bovis Bacillus Calmette-Guerin (BCG) Vaccination in a Cohort of Gambian infants

Background

There is a need for new vaccines for tuberculosis (TB) that protect against adult pulmonary disease in regions where BCG is not effective. However, BCG could remain integral to TB control programmes because neonatal BCG protects against disseminated forms of childhood TB and many new vaccines rely on BCG to prime immunity or are recombinant strains of BCG. Interferon-gamma (IFN-γ) is required for immunity to mycobacteria and used as a marker of immunity when new vaccines are tested. Although BCG is widely given to neonates IFN-γ responses to BCG in this age group are poorly described. Characterisation of IFN-γ responses to BCG is required for interpretation of vaccine immunogenicity study data where BCG is part of the vaccination strategy.

Methodology/Principal Findings

236 healthy Gambian babies were vaccinated with M. bovis BCG at birth. IFN-γ, interleukin (IL)-5 and IL-13 responses to purified protein derivative (PPD), killed Mycobacterium tuberculosis (KMTB), M. tuberculosis short term culture filtrate (STCF) and M. bovis BCG antigen 85 complex (Ag85) were measured in a whole blood assay two months after vaccination. Cytokine responses varied up to 10 log-fold within this population. The majority of infants (89–98% depending on the antigen) made IFN-γ responses and there was significant correlation between IFN-γ responses to the different mycobacterial antigens (Spearman''s coefficient ranged from 0.340 to 0.675, p = 10⁻⁶–10⁻²²). IL-13 and IL-5 responses were generally low and there were more non-responders (33–75%) for these cytokines. Nonetheless, significant correlations were observed for IL-13 and IL-5 responses to different mycobacterial antigens

Conclusions/Significance

Cytokine responses to mycobacterial antigens in BCG-vaccinated infants are heterogeneous and there is significant inter-individual variation. Further studies in large populations of infants are required to identify the factors that determine variation in IFN-γ responses.     

Isolation and Partial Characterization of Escherichia coli Mutants with Low Levels of Transfer Ribonucleic Acid Nucleotidyltransferase

To determine the function of the enzyme transfer ribonucleic acid (tRNA) nucleotidyltransferase in vivo, five mutants of Escherichia coli containing low levels of this enzyme were isolated. Since no selection procedure for such mutants existed, these strains were isolated by assay of large numbers of colonies from a heavily mutagenized stock. A procedure employing cells made permeable to tRNA and ATP was used to screen the large number of colonies required for the isolation. All the mutants contained less than 20% of the normal level of the AMP-incorporating activity of tRNA nucleotidyltransferase in extracts prepared by several methods, and the best mutant contained only about 2% of this activity. Three of the mutants also had equally low levels of the cytidine 5'-monophosphate-incorporating activity of the enzyme. Despite these low activities, the mutant strains displayed relatively normal growth characteristics at all temperatures examined. The enzyme in the mutant strains was not temperature sensitive, nor were any other abnormal biochemical properties detected. tRNA isolated from the mutant strains was missing significant amounts of its 3' terminal adenosine 5'-monophosphate residue, amounting to 10 to 15% in the best mutant. However, only small amounts of the terminal cytidine 5'-monophosphate residue were missing. The results indicate that tRNA nucleotidyltransferase is involved in some aspect of synthesis or repair of the 3' terminus of tRNA, and that the enzyme is present in large excess over its requirements for this function.     

Monoclonal antibodies recognizing cell surface antigens in Drosophila melanogaster

Monoclonal antibodies have been made against cell surface antigens from Drosophila melanogaster, as probes for “differentiation antigens.” The immunogens used were 0–16 hr embryonic cells and mass isolated imaginal discs. The tissue distributions of the antigens recognized by three antiembryonic antibodies and two antiimaginal disc antibodies have been defined by indirect immunofluorescent (IIF) assays on differentiated embryonic cell cultures and on dissected third instar larval organs. These antigens fall into two major categories being either ubiquitous or tissue-limited in distribution. In indirect radioimmunoassays against 12 Drosophila cell lines the antigens showing ubiquitous tissue distributions were detectable on all cell lines whereas the tissue-limited antigens were absent from some cell lines. Such a screen against cell lines therefore provides a straightforward means of identifying antibodies against nonubiquitous antigens. One antibody recognizing a tissue-limited antigen was detected as muscle-specific by IIF assays on differentiated embryonic cell cultures. The second tissue-limited antigen was found to label basement membranes, by IIF assays against third instar larval organs. The temporal distribution of the antigens during embryogenesis (3–21 hr) has been studied by indirect radioimmunoassay. In this respect the antigens fall into three classes: (1) abundant throughout embryogenesis (a ubiquitous antigen), (2) present throughout embryogenesis but increasing markedly in abundance between 12 and 15 hr (two ubiquitous antigens and the muscle-specific antigen), and (3) absent early in development but appearing at about 12 hr postfertilization (the tissue-limited, basement membrane antigen).     

Fluorescent Antibody Study of the Gram-Positive Anaerobic Cocci

Fluorescent antibody conjugates were prepared from five species of anaerobic cocci commonly isolated from human infections. When tested with homologous and heterologous cells these conjugates were found to be highly specific. There was no evidence of a common genus antigen. Peptococcus magnus conjugates detected a species-specific antigen; cross-reactions with Peptostreptococcus anaerobius were readily eliminated by absorption. The conjugates from Peptococcus asaccharolyticus, Peptococcus prevotii, Peptostreptococcus, anaerobius, and Peptostreptococcus intermedius displayed a high degree of strain specificity. Occasional cross-reactions were detected with homologous strains, suggesting the presence of common antigens, but no attempt was made to determine the number of different serotypes in these species.     

Expression of Epstein-Barr viral capsid, complement fixing, and nuclear antigens in stationary and exponential phase cultures

Three continuous lymphoblastoid cell lines, 2 productive of nucleocapsids and 1 nonproductive line, were studied for their content of Epstein-Barr viral (EBV) antigens during transition from stationary to logarithmic phase growth. As a preliminary step, viable cells were separated from degenerating ones in discontinuous gradients of serum albumin. Viral capsid antigens were found in both living and dead cells of the 2 producer lines; however, complement fixing (CF) antigens and nuclear antigen were detected only in viable cell subpopulations. The content of antigen detectable in extracts of viable cells by complement fixation remained constant in replicating and resting cultures; further, all viable cells of the 3 lines demonstrated intranuclear antigen by anticomplement immunofluorescence in all stages of growth. In contrast, the proportion of cells with viral capsid antigen in the producer lines increased 7- to 24-fold following entry of resting populations into the phase of exponential growth.The results suggest that expression of viral capsid antigens is discontinuous and is initiated in response to events in log phase, possibly DNA synthesis or mitosis. Expression of the complement fixing and nuclear antigens in continuous in viable cells. These findings emphasize the intimate relationship of the CF and nuclear antigen to the transformed state and suggest that study of this antigen complex will shed light on the mechanisms of lymphocyte transformation by EBV.     

Production of heat-stable exotoxin by Bacillus thuringiensis and related bacteria

Heat-stable exotoxin production by 740 strains of Bacillus thuringiensis and related bacteria was investigated using the housefly, Musca domestica, from the following viewpoints: (1) the relation-ship between B. thuringiensis flagellar (H) serotypes and exotoxin production and (2) the exotoxin production by Bacillus species other than B. thuringiensis. Of 437 isolates belonging to 11 serotypes of B. thuringiensis which had been confirmed to produce parasporal inclusions, 35 isolates belonging to serotypes 1, 3a:3b, 4a:4c, and 10 produced heat-stable exotoxin. Exotoxin was not detected in the isolates of serotypes 3a, 4a:4b, 5a:5b, 5a:5c, 6, 7, and 8a:8b. No heat-stable exotoxin was demonstrated in 28 acrystalliferous isolates which possessed H antigens of B. thuringiensis serotypes 1, 3a, 4a:4b, 4a:4c, 5a:5c, 6, 7, 10, 11a:11c, and 12. A total of 270 B. cereus isolates which did not possess B. thuringiensis H antigen were examined and three isolates were found to produce heat-stable exotoxin. No heat-stable exotoxin was produced by B. subtilis (two strains), B. natto (one strain), and B. megaterium (two strains). These results indicate that the heat-stable exotoxin production in B. thuringiensis is a strain-specific property rather than a serotype(subspecies)-specific property.     

Polymorphism in the HASPB Repeat Region of East African Leishmania donovani Strains

Background/Objectives

Visceral leishmaniasis (VL) caused by Leishmania donovani is a major health problem in Ethiopia. Parasites in disparate regions are transmitted by different vectors, and cluster in distinctive genotypes. Recently isolated strains from VL and HIV-VL co-infected patients in north and south Ethiopia were characterized as part of a longitudinal study on VL transmission.

Methodology/Principal Findings

Sixty-three L. donovani strains were examined by polymerase chain reaction (PCR) targeting three regions: internal transcribed spacer 1 (ITS1), cysteine protease B (cpb), and HASPB (k26). ITS1- and cpb - PCR identified these strains as L. donovani. Interestingly, the k26 - PCR amplicon size varied depending on the patient''s geographic origin. Most strains from northwestern Ethiopia (36/40) produced a 290 bp product with a minority (4/40) giving a 410 bp amplicon. All of the latter strains were isolated from patients with HIV-VL co-infections, while the former group contained both VL and HIV-VL co-infected patients. Almost all the strains (20/23) from southwestern Ethiopia produced a 450 bp amplicon with smaller products (290 or 360 bp) only observed for three strains. Sudanese strains produced amplicons identical (290 bp) to those found in northwestern Ethiopia; while Kenyan strains gave larger PCR products (500 and 650 bp). High-resolution melt (HRM) analysis distinguished the different PCR products. Sequence analysis showed that the k26 repeat region in L. donovani is comprised of polymorphic 13 and 14 amino acid motifs. The 13 amino acid peptide motifs, prevalent in L. donovani, are rare in L. infantum. The number and order of the repeats in L. donovani varies between geographic regions.

Conclusions/Significance

HASPB repeat region (k26) shows considerable polymorphism among L. donovani strains from different regions in East Africa. This should be taken into account when designing diagnostic assays and vaccines based on this antigen.     

Search for an antibody profile of Rhodococcus equi infection in AIDS patients despite the diversity of isolates and patient immune dysfunction

Diversity of virulence-associated antigens of Rhodococcus equi was detected among thirteen strains isolated from AIDS patients on two continents. One out of four Brazilian isolates presented the virulence-associated antigen of 15- to 17-kDa, and the other three isolates had the 20-kDa virulence-associated antigen. In contrast, only three out of nine Italian isolates were positive for virulence-associated antigens - two for the 15- to 17-kDa antigen and one for the 20-kDa antigen. In four other Italian strains, one or more other low-molecular-weight antigens were identified. Because of R. equi variability and host immune dysfunction, no characteristic antibody profile was detected among patients, although the presence of specific antibodies in serum samples suggested prognostic value: good patient outcome and recovery from pneumonia were correlated with R. equi antibody detection, whereas the lack or disappearance of specific antibodies, mainly those to low-molecular-weight antigens, was correlated with disease progression and patient death. These results confirmed the nonobligatory presence of the well-known virulence-associated antigens for the pathogenicity of R. equi in humans, and also the diversity of R. equi strains isolated from AIDS patients, which may be related to the geographic origin of the isolates or may be a consequence of the route of R. equi transmission in different countries. Some mechanisms underlying the results obtained are discussed, suggesting immune complex formation during the progress of the disease.     

Pooled Protein Immunization for Identification of Cell Surface Antigens in Streptococcus sanguinis

Background

Available bacterial genomes provide opportunities for screening vaccines by reverse vaccinology. Efficient identification of surface antigens is required to reduce time and animal cost in this technology. We developed an approach to identify surface antigens rapidly in Streptococcus sanguinis, a common infective endocarditis causative species.

Methods and Findings

We applied bioinformatics for antigen prediction and pooled antigens for immunization. Forty-seven surface-exposed proteins including 28 lipoproteins and 19 cell wall-anchored proteins were chosen based on computer algorithms and comparative genomic analyses. Eight proteins among these candidates and 2 other proteins were pooled together to immunize rabbits. The antiserum reacted strongly with each protein and with S. sanguinis whole cells. Affinity chromatography was used to purify the antibodies to 9 of the antigen pool components. Competitive ELISA and FACS results indicated that these 9 proteins were exposed on S. sanguinis cell surfaces. The purified antibodies had demonstrable opsonic activity.

Conclusions

The results indicate that immunization with pooled proteins, in combination with affinity purification, and comprehensive immunological assays may facilitate cell surface antigen identification to combat infectious diseases.     

A Novel Malaria Vaccine Candidate Antigen Expressed in Tetrahymena thermophila

Development of effective malaria vaccines is hampered by the problem of producing correctly folded Plasmodium proteins for use as vaccine components. We have investigated the use of a novel ciliate expression system, Tetrahymena thermophila, as a P. falciparum vaccine antigen platform. A synthetic vaccine antigen composed of N-terminal and C-terminal regions of merozoite surface protein-1 (MSP-1) was expressed in Tetrahymena thermophila. The recombinant antigen was secreted into the culture medium and purified by monoclonal antibody (mAb) affinity chromatography. The vaccine was immunogenic in MF1 mice, eliciting high antibody titers against both N- and C-terminal components. Sera from immunized animals reacted strongly with P. falciparum parasites from three antigenically different strains by immunofluorescence assays, confirming that the antibodies produced are able to recognize parasite antigens in their native form. Epitope mapping of serum reactivity with a peptide library derived from all three MSP-1 Block 2 serotypes confirmed that the MSP-1 Block 2 hybrid component of the vaccine had effectively targeted all three serotypes of this polymorphic region of MSP-1. This study has successfully demonstrated the use of Tetrahymena thermophila as a recombinant protein expression platform for the production of malaria vaccine antigens.     

Molecular characterization of the capsular antigens of Pasteurella multocida isolates using multiplex PCR

The use of molecular techniques for detection and characterization of the Pasteurella multocida is very important for rapid and specific detection and characterization of the organism. During the period from 15th February, 2014 to 15th April, 2015, 425 nasopharyngeal swabs and 175 lung and spleen samples were collected and examined by conventional methods, 80 strains (18.82%) of P. multocida were isolated from the calves, sheep and goat with respiratory manifestation. Meanwhile, 77 strains (44%) were isolated from emergency slaughtered animals. All the recovered strains were positive for specific PCR for detection of P. multocida strains previously identified as P. multocida by standard microbiological techniques. Multiplex PCR for molecular typing of the capsular antigens of the recovered P. multocida revealed positive amplification of 1044 bp fragments specific to the capsular antigen type A with 105 strains (66.88%), and amplification 511 bp fragments of the capsular antigen type E with 52 strain (33.12%) and absence of B, D and F antigens. Multiplex PCR for molecular typing of the capsular antigens of P. multocida can be used as a simple, sensitive, rapid, reliable technique instead of the serological techniques for identification of the capsular antigens of P. multocida