Production and characterization of monoclonal antibodies against vegetative cells of Bacillus cereus

Two monoclonal antibodies (MAbs) against Bacillus cereus were produced. The MAbs (8D3 and 9B7) were selected by enzyme-linked immunosorbent assay for their reactivity with B. cereus vegetative cells. They reacted with B. cereus vegetative cells while failing to recognize B. cereus spores. Immunoblotting revealed that MAb 8D3 recognized a 22-kDa antigen, while MAb 9B7 recognized two antigens with molecular masses of approximately 58 and 62 kDa. The use of MAbs 8D3 and 9B7 in combination to develop an immunological method for the detection of B. cereus vegetative cells in foods was investigated.     

Establishment of Monoclonal Antibodies Specific for Bacillus subtilis DB9011

Bacillus subtilis DB9011 is a strain with useful functions for agriculture. To establish a method for the discrimination of this strain from others, monoclonal antibodies (MAbs) were prepared. Although two established MAbs (MAb9B6 and MAb14D2) cross-react with some other Bacillus strains in ELISA, only B. subtilis DB9011 vegetative cells are recognized by both MAbs. MAb14D2 recognizes flagellin, a 34-kDa unit protein of flagella. The two MAbs established will provide powerful tools with which detailed analysis of this bacterial strain can be obtained under environmental conditions.     

Establishment of monoclonal antibodies specific for Bacillus subtilis DB9011

Bacillus subtilis DB9011 is a strain with useful functions for agriculture. To establish a method for the discrimination of this strain from others, monoclonal antibodies (MAbs) were prepared. Although two established MAbs (MAb9B6 and MAb14D2) cross-react with some other Bacillus strains in ELISA, only B. subtilis DB9011 vegetative cells are recognized by both MAbs. MAb14D2 recognizes flagellin, a 34-kDa unit protein of flagella. The two MAbs established will provide powerful tools with which detailed analysis of this bacterial strain can be obtained under environmental conditions.     

Development and Application of Pathovar-Specific Monoclonal Antibodies That Recognize the Lipopolysaccharide O Antigen and the Type IV Fimbriae of Xanthomonas hyacinthi

The objective of this study was to develop a specific immunological diagnostic assay for yellow disease in hyacinths, using monoclonal antibodies (MAbs). Mice were immunized with a crude cell wall preparation (shear fraction) from Xanthomonas hyacinthi and with purified type IV fimbriae. Hybridomas were screened for a positive reaction with X. hyacinthi cells or fimbriae and for a negative reaction with X. translucens pv. graminis or Erwinia carotovora subsp. carotovora. Nine MAbs recognized fimbrial epitopes, as shown by immunoblotting, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy; however, three of these MAbs had weak cross-reactions with two X. translucens pathovars in immunoblotting experiments. Seven MAbs reacted with lipopolysaccharides and yielded a low-mobility ladder pattern on immunoblots. Subsequent analysis of MAb 2E5 showed that it specifically recognized an epitope on the O antigen, which was found to consist of rhamnose and fucose in a 2:1 molar ratio. The cross-reaction of MAb 2E5 with all X. hyacinthi strains tested showed that this O antigen is highly conserved within this species. MAb 1B10 also reacted with lipopolysaccharides. MAbs 2E5 and 1B10 were further tested in ELISA and immunoblotting experiments with cells and extracts from other pathogens. No cross-reaction was found with 27 other Xanthomonas pathovars tested or with 14 other bacterial species from other genera, such as Erwinia and Pseudomonas, indicating the high specificity of these antibodies. MAbs 2E5 and 1B10 were shown to be useful in ELISA for the detection of X. hyacinthi in infected hyacinths.     

Effects of Lactic Acid Bacteria and Organic Acids on Growth and Germination of Bacillus cereus

Growth and germination of vegetative cells and endospores of Bacillus cereus were affected by Streptococcus lactis, Streptococcus thermophilus, Lactobacillus acidophilus, and Lactobacillus bulgaricus in nonfat milk medium and by salts of organic acids in broth medium. Growth of the lactic acid bacteria was not affected by B. cereus. B. cereus increased rapidly to about 10⁸ CFU/ml when cells were added at the beginning of growth of lactic acid bacteria; it was inactivated slowly when added after 24 h and rapidly when added after 72 h of lactic acid bacterial growth. Streptococci were more inhibitory to the growth of B. cereus than lactobacilli were at 24 h. Spore germination was not affected after 24 h, but it was inhibited after 48 and 72 h of lactic acid bacterial growth. Acetate was more inhibitory to the growth of vegetative cells, while formate was more inhibitory to spore germination. Acetate, formate, and lactate (all at 0.1 M) completely inactivated multiplication of B. cereus at pH 6.1, 6.0, and 5.6, respectively. Spores of B. cereus were more resistant to these organic acids compared with the resistance of vegetative cells. Formate, lactate, and acetate (all at 0.1 M) caused 50% inhibition of spore germination at pH 4.4, 4.3, and 4.2, respectively.     

Rapid identification and differentiation of Vibrio parahaemolyticus from Vibrio spp. in seafood samples using developed monoclonal antibodies

Monoclonal antibodies (MAbs) specific to Vibrio parahaemolyticus were successfully generated. According to the specificity of V. parahaemolyticus, MAbs can be classified into 5 groups. The MAbs VP-2D and VP-11H were specific to the O2 and O4 groups of V. parahaemolyticus, respectively. The MAb VP-11B reacted with 11 out of 30 isolates of V. parahaemolyticus used in this study. The MAb VP-516 bound to 27 out of 30 isolates of V. parahaemolyticus and cross reacted with all 10 isolates of V. alginolyticus. The MAb VP-618 demonstrated positive reactivity to 29 out of 30 isolates of V. parahaemolyticus and demonstrated slight cross reactivity to 3 out of 30 isolates of V. harveyi. The sensitivity of the MAbs ranged from 10⁸ to 10⁷ c.f.u. ml^?1 for V. parahaemolyticus obtained from pure cultures and depended on the group of MAbs. However, the detection capability could be improved to be equivalent to that of the PCR technique following pre-incubation of the samples in alkaline peptone water (APW). Using these MAbs along with MAbs specific to V. alginolyticus (VA-165), V. cholerae (VC-63), V. harveyi (VH-9B and VH-20C) and Vibrio spp. (VC-201) from previous studies, V. parahaemolyticus could be identified and differentiated from Vibrio spp. in various seafood samples including shrimp, green mussels, blood clams and oysters by a simple dot blot immunoassay without the requirement for bacterial isolation or biochemical characterization.     

Detection of multiple antigenically related proteins from various rat tissues by monoclonal antibodies against 3 alpha-hydroxysteroid dehydrogenase.

Mouse hybridomas were prepared by fusing myelomas and spleen cells from mice immunized with purified rat 3 alpha-hydroxysteroid dehydrogenase. Hybridomas secreting monoclonal antibodies against 3 alpha-hydroxysteroid dehydrogenase were selected by indirect enzyme-linked immunoassay and then subcloned by limiting dilution. From two mice we have obtained four positive hybridomas, three secreting high affinity immunoglobulin (Ig) G1 and one secreting IgM. Only two of these monoclonal antibodies (MAbs 3G6 and 7D3, both IgG1) recognized denatured enzyme and, therefore, were used for further immunoblotting experiments. MAb 7D3 recognized a structurally related mouse enzyme, but not the human enzyme, whereas monoclonal antibody 3G6 recognized a human enzyme, but not the mouse enzyme. When these two monoclonal antibodies were used in immunoblotting to survey the expression of 3 alpha-hydroxysteroid dehydrogenase in rat liver and a number of other tissues, striking differences were found in the protein band patterns in kidney, lung, and testis. Both MAbs 7D3 and 3G6 recognized 3 alpha-hydroxysteroid dehydrogenase, a 34-kDa 7D3 recognized a protein of the same size as the liver protein, whereas MAb 3G6 recognized a 34-kDa protein plus another protein of 36 kDa. In kidney only MAb 3G6, but not MAb 7D3, recognized a 34-kDa protein. Conversely, the 34-kDa protein in testis was recognized by MAb 7D3, but not by MAb 3G6. These findings suggest the existence of multiple antigenically related proteins in different tissues.     

Monoclonal antibodies to Hammondia hammondi allowing immunological differentiation from Toxoplasma gondii

Five murine monoclonal antibodies (MAbs) were developed against purified sporozoites of Hammondia hammondi. Despite a large antigenic similarity between the 2 closely related coccidia, H. hammondi and Toxoplasma gondii, these MAbs only reacted with H. hammondi. Three MAbs, ID3, 3F2, and 4C9-7, recognized antigens of 38 kDa localized in rhoptries (1D3), in rhoptries and in oocyst and cyst walls (3F2), and in rhoptries and the apical region (4C9-7). Another MAb, 4C9-10, reacted with a 27-kDa antigen in dense granules of sporozoites and tachyzoites, and MAB 11B3 labeled an antigen of >94 kDa located in the pellicular membrane of the 3 stages of the parasite. These MAbs could be used for a rapid discrimination of the 2 coccidia in epidemiological studies or for diagnostic purposes in tissues.     

Antigenic characterization of Brazilian isolates of Anaplasma marginale

Antigenic characterization of Anaplasma marginale isolates, by identifying conserved and variable epitopes of major surface proteins (MSP), is an important tool for vaccine development against this rickettsia. The B cell epitopes of A. marginale isolates from three microregions of the State of Pernambuco and one from the State of Mato Grosso do Sul, Brazil, were characterized by indirect fluorescent antibody technique (IFAT) and Western blot (WB) with 15 monoclonal antibodies (MAbs). The epitope recognized by MAb ANA22B1 (MSP-1a) was conserved by IFAT and WB (73-81 kDa). MSP-2 epitopes recognized by MAbs ANAO58A2 and ANAO70A2 were conserved by IFAT, while ANAO50A2 and ANA66A2 epitopes were polymorphic; in the WB, the MAbs ANAO50A2 and ANAO70A2 identified bands of 45 kDa only in the Pernambuco-Mata isolate. None of the isolates reacted with MAb ANAR75C2 (MSP-3). The MSP-4 epitope recognized by MAb ANAR76A1 was conserved by IFAT, as well as the MSP-5 epitope recognized by MAb ANAF16C1 by IFAT and WB (16 kDa). The MAbs ANAR17A6, ANAR83B3, ANAR94C1, ANAO24D5 and ANAR19A6 identified conserved epitopes by IFAT. MSP-1, MSP-2 and MSP-4, which previously showed partial protection in experimental trials, are also potential immunogens to be employed in Brazil, due to the B cell epitope conservation.     

Characterization of human serotype 1 astrovirus-neutralizing epitopes.

Astroviruses are important agents of pediatric gastroenteritis. To better understand astrovirus antigenic structure and the basis of protective immunity, monoclonal antibodies (MAbs) were produced against serotype 1 human astrovirus. Four MAbs were generated. One MAb (8G4) was nonneutralizing but reacted to all seven serotypes of astrovirus by enzyme-linked immunosorbentassay (ELISA) and immunoperoxidase staining of infected cells. Three MAbs were found to have potent neutralizing activity against astrovirus. The first (5B7) was serotype 1 specific, another (7C2) neutralized all seven human astrovirus serotypes, while the third (3B2) neutralized serotypes 1 and 7. Immunoprecipitation of radiolabeled astrovirus proteins from supernatants of astrovirus-infected cells showed that all three neutralizing antibodies reacted with VP29. MAb 5B7 also reacted strongly with VP26. A competition ELISA showed that all three neutralizing antibodies competed with each other for binding to purified astrovirus virions, suggesting that their epitopes were topographically in close proximity. None of the neutralizing MAbs competed with nonneutralizing MAb 8G4. The neutralizing MAbs were used to select antigenic variant astroviruses, which were then studied in neutralization assays. These assays also suggested a close relationship between the respective epitopes. All three neutralizing MAbs were able to prevent attachment of radiolabeled astrovirus particles to human Caco 2 intestinal cell monolayers. Taken together, these data suggest that the astrovirus capsid protein VP29 may be important in viral neutralization, heterotypic immunity, and virus attachment to target cells.     

Production and Characterization of Two Monoclonal Antibodies Specific for Plasmopara halstedii

Sunflower downy mildew, caused by the fungus Plasmopara halstedii, is a potentially devastating disease. We produced two monoclonal antibodies (MAbs) (12C9 and 18E2) by immunizing mice with a partially purified extract of P. halstedii race 1. Both MAbs detected in enzyme-linked immunosorbent assay (ELISA) all races of P. halstedii present in France. No cross-reactions were observed with Plasmopara viticola or with other fungi commonly associated with sunflowers. Both MAbs recognized the same three fungal antigens with molecular masses of 68, 140, and 192 kDa. However, the epitopes on the fungal antigens were distinct and repetitive. Seed homogenates from infected plants were incubated in wells coated with MAb 18E2. This resulted in the trapping of P. halstedii antigens that were identified with biotinylated MAb 12C9. No reactions were seen with seed homogenates from healthy plants. Thus, our results suggest that these MAbs might be used to develop a sandwich ELISA detection system for P. halstedii in infected seeds.     

Critical epitopes in transmissible gastroenteritis virus neutralization.

Purified transmissible gastroenteritis (TGE) virus was found to be composed of three major structural proteins having relative molecular weights of 200,000, 48,000, and 28,000. The peplomer glycoprotein was purified by affinity chromatography with the monoclonal antibody (MAb) 1D.G3. A collection of 48 MAbs against TGE virus was developed from which 26, 10, and 3 were specific for proteins E2, N, and E1, respectively. A total of 14 neutralizing MAbs of known reactivity were E2 protein specific. In addition, MAb 1B.C11, of unknown specificity, was also neutralizing. These MAbs reduced the virus titer 10(2)- to 10(9)-fold. Six different epitopes critical in TGE virus neutralization were found, all of which were conformational based on their immunogenicity and antigenicity. Only the epitope defined by MAb 1G.A7 was resistant to sodium dodecyl sulfate treatment, although it was destroyed by incubation in the presence of both the detergent and beta-mercaptoethanol. The frequency of MAb-resistant (mar) mutants selected with four MAbs (1G.A7, 1B.C11, 1G.A6, and 1E.F9) ranged from 10(-6) to 10(-7), whereas the frequency of the putative mar mutant defined by MAb 1B.B11 was lower than 10(-9). Furthermore, the epitopes defined by these MAbs and by MAbs 1H.C2 and 1A.F10, were present in 11 viral isolated with different geographical locations, years of isolation, and passage numbers (with the exception of two epitopes absent or modified in the TOY 56 viral isolate), suggesting that the critical epitopes in TGE virus neutralization were highly conserved.     

Identification of a Novel Haemophilus parasuis-Specific B Cell Epitope Using Monoclonal Antibody against the OppA Protein

Monoclonal antibody (MAb) 1B3 against Haemophilus parasuis (H. parasuis) was generated by fusing SP2/0 murine myeloma cells and spleen cells from BALB/c mice immunized with the whole-bacterial-cell suspension of H. parasuis HS80 (serotype 5). The MAb 1B3 showed strong reactivity with 15 serotype reference strains of H. parasuis using Dot blot and Western blot analysis. Immunoprecipitation and protein spectral analysis indicated that MAb 1B3 recognized by Oligopeptide permease A (OppA) belongs to the ATP binding cassette transporter family. In addition, a linear B-cell epitope recognized by MAb 1B3 was identified by the screening of a phage-displayed 12-mer random peptide library. Sequence analysis showed that MAb 1B3 was recognized by phages-displaying peptides with the consensus motif KTPSEXR (X means variable amino acids). Its amino acid sequence matched ⁴⁶⁹KTPAEAR⁴⁷⁵ of H. parasuis OppA protein. A series of progressively truncated peptides were synthesized to define the minimal region that was required for MAb 1B3 binding. The epitope was highly conserved in OppA protein sequences from the isolated H. parasuis strains, which was confirmed by alignment analysis. Furthermore, the minimal linear epitope was highly specific among 75 different bacterial strains as shown in sequence alignments. These results indicated MAb 1B3 might be potentially used to develop serological diagnostic tools for H. parasuis.     

Inhibition of Epstein-Barr virus (EBV) release from P3HR-1 and B95-8 cell lines by monoclonal antibodies to EBV membrane antigen gp350/220.

Antibody-mediated inhibition of Epstein-Barr virus (EBV) release from the EBV-productive cell lines P3HR-1 and B95-8 was probed with two monoclonal antibodies (MAbs), 72A1 and 2L10, which immunoprecipitated the same EBV membrane antigen (MA) gp350/220 found with the 1B6 MAb with which inhibition of EBV release from P3HR-1 cells was first described. These three MAbs were not equivalent in either MA reactivities or functional effects, reflecting the variable expression of different epitopes of gp350/220. 1B6 recognized MA on P3HR-1 cells, which expressed predominately the gp220 form of MA. 1B6 did not recognize (or barely recognized) a determinant on B95-8 cells. MAbs 2L10 and 72A1 reacted as well with B95-8 cells as they did with P3HR-1 cells. MAbs 1B6 and 2L10 neutralized neither P3HR-1 nor B95-8 virus, but 72A1 neutralized both viruses. MAbs 1B6 and 72A1 inhibited P3HR-1 virus release, as measured by the assay for infectious virus and by DNA hybridization analysis of released virus, but 2L10 had no such activity. 72A1 (but not 1B6) inhibited release of EBV from B95-8 cells. These experiments pointed to the presence of three different epitopes on gp350/220, identified with the respective MAbs and having varying involvement in virus neutralization and virus release inhibition.     

Acanthamoeba castellanii: characterization of an adhesin molecule.

Acanthamoeba castellanii is a free-living protozoan that causes keratitis in humans and has been associated with pneumonia and granulomatous amebic encephalitis in dogs, sheep, and other species. Adherence of the Acanthamoeba to epithelial cells is critical to the pathogenesis of this disease. In this study, several mouse monoclonal antibodies (MAb) generated to whole Acanthamoeba trophozoites identified surface membrane epitopes by ELISA and IFA. Nine antibodies inhibited adherence of [(35)S]-methionine-labeled Acanthamoeba trophozoites to hamster corneal epithelial cells by 27-90%. Sodium periodate treatment, but not proteinase K digestion, of whole Acanthamoeba destroyed epitopes recognized by adherence-inhibiting antibodies such as MAb 7H6, suggesting that the adherence epitopes are carbohydrates. Other antibodies, MAb 2A8 for example, recognized surface membrane peptide epitopes that were proteinase K sensitive and sodium periodate resistant. Purified MAb 2A8 was used in an antigen-capture ELISA with peroxidase-labeled MAb 7H6 and demonstrated that the carbohydrate adhesion molecule was linked to the peptide recognized by MAb 2A8. Both MAbs 7H6 and 2A8 recognized a >207-kDa band on a Western blot of eluant from a MAb 2A8 immunoaffinity column, confirming that MAb 7H6 and MAb 2A8 recognize different epitopes on the same adherence molecule. MAbs 7H6 and 2A8 also identified the adhesion molecule in soluble Acanthamoeba membrane preparations and MAb 2A8 immunoaffinity column eluant by ELISA and Western blot. Neither of these antibodies were inhibited from binding to whole trophozoites nor membrane extracts by mannose or mannan in competitive binding assays. When our Acanthamoeba membrane preparations were electrophoresed and immunoblotted with alpha-d-mannosylated-biotin albumin, no bands were recognized in the >207 kDa range by our adherence-associated antibodies. These results suggest that the Acanthamoeba adhesin is not identical to the mannose binding protein of Acanthamoeba but rather is a distinct surface membrane glycoprotein.     

Comparative biodistribution studies of DTPA-derivative bifunctional chelates for radiometal labeled monoclonal antibodies

Biodistribution of five different backbone-substituted derivatives of SCN-Bz-DTPA (1B4M-DTPA, 1M3B-DTPA, 1B3M-DTPA, GEM-DTPA and 2B-DTPA) linked to MAb B72.3 were compared to that of the parent molecule after labeling with ¹¹¹indium. Athymic mice, bearing human colon carcinoma xenografts (LS-174T) were injected i.v. to determine the biodistribution of the MAb chelate conjugates. Three of the MAb metal chelate conjugates (1B4M-DTPA, 1M3B-DTPA, and 1B3M-DTPA), labeled with ¹¹¹In showed efficient and stable tumor localization as well as a slower blood clearance rate than SCN-Bz-DTPA, GEM-DTPA or 2B-DTPA MAb chelate conjugates. Major differences were also seen in normal organ uptake, especially liver and spleen. Tumor-to-liver ratios rose as a function of time for 1B4M-DTPA, 1M3B-DTPA and 1B3M-DTPA MAb chelate conjugates with virtually no accumulation of the radiometal into this organ, as revealed by no increase in the liver-to-blood values. Small accretion in normal liver was noted for SCN-Bz-DTPA, GEM-DTPA or 2B-DTPA MAb chelate conjugates. The results reviewed here, and described previously (Roselli et al., 1991) demonstrate that the use in vivo of backbone-substituted forms of the SCN-Bz-DTPA, such as 1B4M-DTPA, 1M3B-DTPA, and 1B3M-DTPA bound to MAbs, can reduce uptake of indium to normal organs while maximizing the dose to tumor.     

Identification of a Highly Conserved Epitope on Avian Influenza Virus Non-Structural Protein 1 Using a Peptide Microarray

Avian influenza virus (AIV) non-structural protein 1 (NS1) is a multifunctional protein. It is present at high levels in infected cells and can be used for AIV detection and diagnosis. In this study, we generated monoclonal antibody (MAb) D7 against AIV NS1 protein by immunization of BALB/c mice with purified recombinant NS1 protein expressed in Escherichia coli. Isotype determination revealed that the MAb was IgG₁/κ-type subclass. To identify the epitope of the MAb D7, the NS1 protein was truncated into a total of 225 15-mer peptides with 14 amino acid overlaps, which were spotted for a peptide microarray. The results revealed that the MAb D7 recognized the consensus DAPF motif. Furthermore, the AIV NS1 protein with the DAPF motif deletion was transiently expressed in 293T cells and failed to react with MAb D7. Subsequently, the DAPF motif was synthesized with an elongated GSGS linker at both the C- and N-termini. The MAb D7 reacted with the synthesized peptide both in enzyme-linked immunosorbent assay (ELISA) and dot-blot assays. From these results, we concluded that DAPF motif is the epitope of MAb D7. To our knowledge, this is the first report of a 4-mer epitope on the NS1 protein of AIV that can be recognized by MAb using a peptide microarray, which is able to simplify epitope identification, and that could serve as the basis for immune responses against avian influenza.     

Role for β2-Microglobulin in Echovirus Infection of Rhabdomyosarcoma Cells

A monoclonal antibody (MAb) that blocks most echoviruses (EVs) from infecting rhabdomyosarcoma (RD) cells has been isolated. By using the CELICS cloning method (T. Ward, P. A. Pipkin, N. A. Clarkson, D. M. Stone, P. D. Minor, and J. W. Almond, EMBO J. 13:5070–5074, 1994), the ligand for this antibody has been identified as β2-microglobulin (β2m), the 12-kDa protein that associates with class I heavy chains to form class I HLA complexes. A commercial MAb (MAb 1350) against β2m was also found to block EV7 infection without affecting binding to its receptor, DAF, or replication of EV7 viral RNA inside cells. Entry of EV7 into cells was reduced by only 30% by antibody and cytochalasin D, an inhibitor of endocytosis mediated by caveolae and clathrin-coated pits, but was not significantly reduced by sodium azide. The block to virus entry by cytochalasin D was additive to the block induced by antibody. We suggest that EV7 rapidly enters into a multicomponent receptor complex prior to entry into cells and that this initial entry event requires β2m or class I HLA for infection to proceed.Echoviruses (EVs) are members of the Enterovirus genus of the family Picornaviridae and are important human pathogens. They are associated with a wide spectrum of clinical syndromes, including rashes, diarrhea, aseptic meningitis, respiratory disease, and possibly conditions such as chronic fatigue syndrome. This range of clinical manifestations is probably a reflection of virus tissue tropisms, which seem to be mediated, at least in part, by utilization of a range of cellular receptors.Anti-cell surface monoclonal antibodies (MAbs) that block EV infection have been isolated previously and have been used to determine the identity of some of these receptors. In 1992 Bergelson et al. demonstrated that EV serotypes 1 and 8 use the collagen receptor VLA-2 (6) by attaching to the α2 subunit (7). Previously, we and others have shown that a regulator of complement activity, decay-accelerating factor (DAF), is the receptor for a range of hemagglutinating EVs (3, 37). Other EVs appear to use neither of these, but the identity of their receptor(s) is unknown. Mbida et al. have isolated a MAb (MAb 143) that blocks most EV serotypes from infecting a range of cell types. MAb 143 was also found to block coxsackievirus A9 but not poliovirus or coxsackievirus serotypes B1 to B6 (21). The ligand for MAb 143 was found by affinity purification to be an unknown 44-kDa glycoprotein (22). It was therefore suggested that the 44-kDa protein was part of a multicomponent receptor complex used by most EVs to infect cells. A direct role for the 44-kDa protein in virus attachment seems unlikely, since MAb 143 blocks infection by the viruses that have been shown to use other proteins, such as DAF (3, 37) and VLA-2 (6), as their primary receptors.Here, we report the isolation of a MAb similar to that described by Mbida et al. (21, 22) and describe the cloning and identification of its ligand. The ligand is β2-microglobulin (β2m), a 12-kDa protein that associates with the class I HLA heavy chains (44 kDa) and presents antigenic peptides (20). We show that MAbs to β2m block EV infection partly by reducing the entry of virus into cells, although other postbinding effects cannot be ruled out.     

Germination and proliferation of emetic Bacillus cereus sensu lato strains in milk

The Bacillus cereus sensu lato group includes potentially pathogenic bacteria that are ubiquitous in the environment and, importantly, could also be present in food products. This study focuses on emetic isolates which presumably could cause acute food poisoning and emetic syndrome. Here, we evaluate the ability of psychrotolerant Bacillus weihenstephanensis MC118 (isolated from soil) and mesophilic B. cereus BOD3/9 isolated from milk to germinate and multiply at 7 and 30 °C. Whereas the rates of germination at 30 °C in milk and nutrient broth of MC118 and BOD3/9 were similar, MC118, but not BOD3/9, proliferated to achieve relatively high numbers (～10⁶?colony-forming units/g) within 7 days of incubation at 7 °C. Mesophilic BOD3/9 showed a slight decrease of cell concentration in similar studies at 7 °C. Genotyping with repetitive extragenic palindromic sequence-based PCR and pulsed field gel electrophoresis revealed significant similarities between BOD3/9 and emetic reference B. cereus F4810/72 strain, while the B. weihenstephanensis MC118 isolate was more similar to the B. weihenstephanensis non-emetic reference DSMZ11821 strain. Our data suggest that emetic isolates that are also psychrotolerant, such as MC118, could constitute a hazard in the dairy industry, where milk could be a suitable medium for germination and growth.