Identification and characterization of a DNA region involved in the export of capsular polysaccharide by Actinobacillus pleuropneumoniae serotype 5a

Actinobacillus pleuropneumoniae synthesizes a serotype-specific capsular polysaccharide that acts as a protective barrier to phagocytosis and complement-mediated killing. To begin understanding the role of A. pleuropneumoniae capsule in virulence, we sought to identify the genes involved in capsular polysaccharide export and biosynthesis. A 5.3-kb XbaI fragment of A. pleuropneumoniae serotype 5a J45 genomic DNA that hybridized with DNA probes specific for the Haemophilus influenzae type b cap export region was cloned and sequenced. This A. pleuropneumoniae DNA fragment encoded four open reading frames, designated cpxDCBA. The nucleotide and predicted amino acid sequences of cpxDCBA contained a high degree of homology to the capsule export genes of H. influenzae type b bexDCBA, Neisseria meningitidis group B ctrABCD, and, to a lesser extent, Escherichia coli K1 and K5 kpsE and kpsMT. When present in trans, the cpxDCBA gene cluster complemented kpsM::TnphoA or kpsT::TnphoA mutations, determined by enzyme immunoassay and by restored sensitivity to a K5-specific bacteriophage. A cpxCB probe hybridized to genomic DNA from all A. pleuropneumoniae serotypes tested, indicating that this DNA was conserved among serotypes. These data suggest that A. pleuropneumoniae produces a group II family capsule similar to those of related mucosal pathogens.     

Cloning and mutagenesis of a serotype-specific DNA region involved in encapsulation and virulence of Actinobacillus pleuropneumoniae serotype 5a: concomitant expression of serotype 5a and 1 capsular polysaccharides in recombinant A. pleuropneumoniae serotype 1

A DNA region involved in Actinobacillus pleuropneumoniae serotype 5 capsular polysaccharide (CP) biosynthesis was identified and characterized by using a probe specific for the cpxD gene involved in CP export. The adjacent serotype 5-specific CP biosynthesis region was cloned from a 5.8-kb BamHI fragment and an 8.0-kb EcoRI fragment of strain J45 genomic DNA. DNA sequence analysis demonstrated that this region contained four complete open reading frames, cps5A, cps5B, cps5C, and cps5D. Cps5A, Cps5B, and Cps5C showed low homology with several bacterial glycosyltransferases involved in the biosynthesis of lipopolysaccharide or CP. However, Cps5D had high homology with KdsA proteins (3-deoxy-D-manno-2-octulosonic acid 8-phosphate synthetase) from other gram-negative bacteria. The G+C content of cps5ABC was substantially lower (28%) than that of cps5D and the rest of the A. pleuropneumoniae chromosome (42%). A 2.1-kb deletion spanning the cloned cps5ABC open reading frames was constructed and transferred into the J45 chromosome by homologous recombination with a kanamycin resistance cassette to produce mutant J45-100. Multiplex PCR confirmed the deletion in this region of J45-100 DNA. J45-100 did not produce intracellular or extracellular CP, indicating that cps5A, cps5B, and/or cps5C were involved in CP biosynthesis. However, biosynthesis of the Apx toxins, lipopolysaccharide, and membrane proteins was unaffected by the mutation. Besides lack of CP biosynthesis, and in contrast to J45, J45-100 grew faster, was sensitive to killing in precolostral calf serum, and was avirulent in pigs at an intratracheal challenge dose three times the 50% lethal dose (LD50) of strain J45. At six times the J45 LD50, J45-100 caused mild to moderate lung lesions but not death. Electroporation of cps5ABC into A. pleuropneumoniae serotype 1 strain 4074 generated strain 4074(pJMLCPS5), which expressed both serotype 1 and serotype 5 CP. However, serotype 1 capsule expression was diminished in 4074(pJMLCPS5) in comparison to 4074. The recombinant strain produced significantly less total CP (serotypes 1 and 5 CP combined) in log phase (P = 0.0012) but significantly more total CP in late stationary phase than 4074 (P < 0.0001). In addition, strain 4074(pJMLCPS5) caused less mortality and bacteremia in pigs and mice following respiratory challenge than strain 4074, indicating that virulence was affected by diminished capsule production. These results emphasize the importance of CP in the serum resistance and virulence of A. pleuropneumoniae.     

Genetic analysis of type 5 capsular polysaccharide expression by Staphylococcus aureus

Capsules are produced by over 90% of Staphylococcus aureus strains, and approximately 25% of clinical isolates express type 5 capsular polysaccharide (CP5). We mutagenized the type 5 strain Reynolds with Tn918 to target genes involved in CP5 expression. From a capsule-deficient mutant, we cloned into a cosmid vector an approximately 26-kb EcoRI fragment containing the transposon insertion. In the absence of tetracycline selection, Tn918 was spontaneously excised, thereby resulting in a plasmid containing 9.4 kb of S. aureus DNA flanking the Tn918 insertion site. The 9.4-kb DNA fragment was used to screen a cosmid library prepared from the wild-type strain. Positive colonies were identified by colony hybridization, and a restriction map of one clone (pJCL19 with an approximately 34-kb insert) carrying the putative capsule gene region was constructed. Fragments of pJCL19 were used to probe genomic DNA digests from S. aureus strains of different capsular serotypes. Fragments on the ends of the cloned DNA hybridized to fragments of similar sizes in most of the strains examined. Blots hybridized to two fragments flanking the central region of the cloned DNA showed restriction fragment length polymorphism. A centrally located DNA fragment hybridized only to DNA from capsular types 2, 4, and 5. DNA from pJCL19 was subcloned to a shuttle vector for complementation studies. A 6.2-kb EcoRI-ClaI fragment complemented CP5 expression in a capsule-negative mutant derived by mutagenesis with ethyl methanesulfonate. These experiments provide the necessary groundwork for identifying genes involved in CP5 expression by S. aureus.     

Detection of an Actinobacillus pleuropneumoniae serotype 2 lipopolysaccharide (LPS) variant

Until now 12 serotypes of Actinobacillus pleuropneumoniae have been recognized. The specificity of the serotypes reside in the carbohydrate composition of the capsular polysaccharides and lipopolysaccharides (LPS). The LPS of A. pleuropneumoniae serotype 2 is a smooth type LPS with O-chains of linear repeating pentasaccharide units with an O-acetyl group linked to a glucose unit. A monoclonal antibody (MAb 102-G02) directed against A. pleuropneumoniae serotype 2 was characterized in enzyme linked immunosorbent assay (ELISA) and in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The MAb 102-G02 was directed against an epitope on the O-chain of the LPS and was used to define a new. LPS variant of A. pleuropneumoniae serotype 2 (referred to as A. pleuropneumoniae serotype 2X). Investigation of the reactivity of the MAb 102-G02 against an A. pleuropneumoniae serotype 2X, field isolate (9008) and the Danish App-2 strain 4226 in electron microscopy, confirmed the different binding patterns.     

Hepatitis C virus serotype-specific core and NS4 antibodies in injecting drug users participating in the Amsterdam cohort studies

In the present study, the RIBA HCV serotyping SIA was evaluated with a cohort of injecting drug users. Serotyping may be a rapid and cost-effective method of determining genotypes in cohort studies. In this study, hepatitis C virus (HCV) antibody-positive sera from a cohort of 331 chronically infected injecting drug users, of which 167 were coinfected with human immunodeficiency virus (HIV), were serotyped by the RIBA HCV Serotyping SIA. Among the 331 specimens, serotype-specific antibodies were detected in 250 (sensitivity, 75. 5%), in which serotype 1 was predominant (57.2%), followed by serotype 3 (26.8%). Among the 331 specimens, 164 were HIV negative, and serotype-specific antibodies were detected in 151 (sensitivity, 92.1%), in which serotype 1 was predominant (59.6%), followed by serotype 3 (33.8%). For a subset of 58 samples taken from 19 chronically infected HCV seroconverters with a mean follow-up of 5 years, serotypes were compared with genotypes, which were determined by a line probe assay (HCV LiPa) and by direct sequencing of the products obtained by nested PCR of the 5' untranslated region. Among the 58 samples with known genotypes, serotype-specific antibodies were detected in 38 (total sensitivity, 65.5%), with a specificity of 78.9%. Thirty of these serotypeable samples revealed a serotype that corresponded to the genotype in the 58 samples (total positive predictive value, 51.7%). Of the 58 samples, 23 were coinfected with HIV, and when these were excluded, the total sensitivity increased to 76.5%, with a total specificity of 80.8% and a total positive predictive value of 61.8%. The serotyping assay showed a high total sensitivity (96.3%) for samples positive by HCV RIBA, version 3.0, with four bands. We conclude that the sensitivity of the RIBA HCV serotyping SIA is limited by the immunocompetence of the HCV-infected host. In general, samples from HIV-negative individuals containing genotype 1a had higher sensitivity, specificity, and concordance in the serotyping assay compared with genotyping, whereas samples containing genotype 3a were found to be more cross-reactive and untypeable. Therefore, the prevalence of genotypes other than genotype 1 could be underestimated if they are determined by serotyping, and improvements in specificity are recommended.     

Detection and characterization of the fimA gene of Escherichia coli O157:H7

An expected 850-bp DNA fragment containing fimA, the structural gene for type 1 fimbriae, and flanking sequences was amplified from 39 (of 46) pathogenic and commensal strains of Escherichia coli using the polymerase chain reaction (PCR). Restriction fragment length polymorphism (RFLP) analysis of the amplified products showed 13 HinP1 and four Sau961 restriction profiles among these 39 E. coli strains, revealing the polymorphic nature of this allele. A unique RFLP pattern was shared by E. coli O157:H7, O157:H- and a few O55 serotype strains. DNA sequence analysis of the fimA region demonstrated that E. coli O157:H7 strain 933 and O157:H- strain E32511 contained identical DNA sequences that were distinct from other E. coli strains, especially a 16-bp sequence 5' to fimA that was conspicuously absent only in E. coli O157 strains. Exploiting these differences, a PCR assay was developed that amplifies a 936-bp fragment from all E. coli O157:H7 strains examined to date. This PCR assay offers a simple, rapid, and reliable means to detect E. coli strains of the O157:H7 serotype.     

Serotype H5a5b is a major clone within mosquito-pathogenic strains of Bacillus sphaericus

Seventy six mosquito pathogenic strains of Bacillus sphaericus and 10 non-pathogens were examined by pulsed field gel electrophoresis (PFGE) of SmaI-digested chromosomal DNA. Non-pathogenic strains were clearly distinguished from the entomopathogenic types which were assigned to 21 groups (SmaI restriction patterns; SRPs). Some agreement between SRP based on PFGE and serotyping was noted, in particular all 39 strains of serotype 5a5b examined revealed identical SRPs indicating total conservation of the SmaI restriction site in these bacteria. Serotype 5a5b (SRP 12) strains comprise a widely distributed and abundant clonal lineage. Most serotypes, however, were divided into several SRPs. Seven strains from serotype 2a2b were covered in five SRPs in which toxin synthesis was correlated with chromosomal structure. Similarly, toxicity correlated with SRP in strains from serotypes 3 and 6.     

Evaluation of a novel serotyping system for hepatitis C virus: strong correlation with standard genotyping methodologies

Direct sequencing and analysis of viral genomes are definitive methods for identifying various hepatitis C virus (HCV) genotypes. However, HCV genome sequencing methods are cumbersome and unsuitable for analyzing large numbers of clinical samples. We have developed a convenient, reliable, and reproducible RIBA strip immunoblot assay system for determining HCV serotype. Briefly, the assay consists of an immunoblot strip on which there are five lanes of immobilized serotype-specific HCV peptides from the nonstructural (NS-4) and core regions of the genomes of HCV types 1,2, and 3. HCV serotype is deduced by determining the greatest intensity of reactivity to the NS-4 serotype-specific HCV peptide band in relation to the intensity of the human immunoglobulin G internal control bands on each strip. HCV core peptide reactivity is used only in the absence of NS-4 reactivity. We used this assay to successfully serotype a high percentage of sera from well-documented HCV-infected patients. Our serotyping results correlated 99% with the findings from the standard restriction fragment length polymorphism genotyping methods. Less than 5% of the serum samples were untypeable. For a selected group of alpha interferon-treated patients we observed that the nonresponders (76.2%) and a majority of the responders who relapsed (72.2%) had type 2 HCV infection. A small population (n= 8) of complete responders was split 3:4:1 as type 1, type 2, and type 3, respectively. Our data indicate that this new serotyping assay has the potential to be a highly specific and reliable method for typing of HCV infection in patients.     

Identification by polymerase chain reaction fingerprinting of Cryptococcus neoformans serotype AD

Seventy-three Cryptococcus neoformans isolates and eight other yeast strains were studied. Fingerprints produced by priming with (GACA)4 differentiated C. neoformans from all other yeasts tested and identified the five C. neoformans serotypes. Four major bands of molecular size 800, 540, 475 and 410 bp were recognized for serotypes A, AD and D. Two of them were specific for serotype A and the other two for serotype D isolates. Serotype AD strains were identified by five different genotypic patterns in which at least one of the two bands specific for serotype A and D were present in different combinations. On repeated and simultaneously performed genotype and serotype testing of nine strains, the genotypic pattern did not change, whereas serotyping was unstable in three cases. PCR-fingerprinting using (GACA)4 as a primer proved more stable than serology in discriminating among C. neoformans serotypes A, D and AD and was able to distinguish among serotype AD strains.     

Mechanisms of tumor-induced hypoglycemia with intraabdominal hemangiopericytoma

To evaluate the genetic diversity and relationships in a collection of 85 Danish strains of Streptococcus agalactiae (group B streptococcus) we have performed restriction fragment length polymorphism analysis on EcoRI- and MspI-digested whole-cell DNA using as probes rRNA, DNA fragments representing the genes encoding hyaluronidase, C5a-peptidase, alpha-antigen, and beta-antigen as well as two randomly selected genomic DNA fragments for which the coding potential is unknown. In addition, we have assayed for expression of hyaluronidase activity and beta-antigen. Combined analyses of our data and those previously obtained by multilocus enzyme electrophoresis and serotyping revealed a population separating into six major lineages that correlate with individual serotypes. The significant linkage disequilibrium of alleles indicates that the S. agalactiae population examined is predominantly clonal. Notably, strains expressing the serotype III capsule divide into two distant evolutionary lineages, of which one lacks expression of hyaluronidase activity. Six North American isolates of serotype III clustered together with multiple Danish serotype III strains, showing that the combinations of characters on which the phylogenetic tree was based are conserved worldwide. Occurrence of beta-antigen correlated with a specific version of the alpha-antigen gene and was exclusively associated with a single major phylogenetic lineage. Comparisons with the clinical history of the strains revealed no evidence of differences in pathogenic potential among the six major genetic divisions.     

Incorporation of 15N from ammonium into the N-linked oligosaccharides of an immunoadhesin glycoprotein expressed in Chinese hamster ovary cells

Thirty-six isolates, from man or swine, of Pasteurella multocida subsp. multocida producing (n = 13) or not producing (n = 23) the dermonecrotic toxin (DNT) were studied by numerical analysis, capsular typing and ribotyping. Toxigenic strains were also characterised by restriction fragment length polymorphism (RFLP) of the toxA gene and pulsed-field gel electrophoresis (PFGE). Numerical analysis differentiated the Pasteurella species and subspecies, but did not discriminate between toxigenic and nontoxigenic strains. RFLP demonstrated that toxA was located in a conserved part of the chromosome of all toxigenic strains. Ribotyping provided evidence of a close association between DNT production and one of the six EcoRI ribotypes designated as E2. In contrast, PFGE provided evidence for significant DNA polymorphism amongst the toxigenic strains. Results of phenotypic and genotypic studies suggested that toxigenic strains do not form a clone within the subspecies multocida. No difference was found between toxigenic strains of porcine or human origin by biochemical characterisation, capsular serotyping or genomic typing methods.     

Differentiation of Actinobacillus pleuropneumoniae strains by sequence analysis of 16S rDNA and ribosomal intergenic regions, and development of a species specific oligonucleotide for in situ detection

The aims of this study were to characterize and determine intraspecies and interspecies relatedness of Actinobacillus pleuropneumoniae to Actinobacillus lignieresii and Actinobacillus suis by sequence analysis of the ribosomal operon and to find a species-specific area for in situ detection of A. pleuropneumoniae. Amplification and sequence analysis of the 16S-23S rDNA ribosomal intergenic sequence (RIS) from the three species showed the existence of two RIS's, differing by about 100 bp. Both sequences contained a region resembling the ribonuclease III cleavage site found in Escherichia coli. The smaller RIS contained a Glu-tRNA gene, and the larger one contained genes encoding Ile-tRNA and Ala-tRNA. These tRNA's showed a high sequence homology to the respective tRNA genes found in E. coli. Sequence analysis of the RIS's showed a high degree of genetic similarity of 24 strains of A. pleuropneumoniae. The larger RIS's were different between the 3 species tested. The sequence of the 16S ribosomal gene was determined for 8 serotypes of A. pleuropneumoniae. These sequences showed only minor base differences, indicating a close genetic relatedness of these serotypes within the species. An oligonucleotide DNA probe designed from the 16S rRNA gene sequence of A. pleuropneumoniae was specific for all strains of the target species and did not cross react with A. lignieresii, the closest known relative of A. pleuropneumoniae. This species-specific DNA probe labeled with fluorescein was used for in situ hybridization experiments to detect A. pleuropneumoniae in biopsies of diseased porcine lungs.     

Development of a fluorescent focus identification assay using serotype-specific monoclonal antibodies for detection and quantitation of rotaviruses in a tetravalent rotavirus vaccine

A fluorescent focus identification assay (FFIDA) was developed for use in experimental studies and for quantitation of the components in a tetravalent live oral rotavirus vaccine. The assay utilizes four serotype-specific neutralizing monoclonal antibodies (MAb) to detect and quantify individual rotaviruses by immunofluorescence staining of fixed virus-infected monkey kidney cells. In mixed virus infections, all four MAb, W1 (serotype 1), 1C10 (serotype 2), R1 (serotype 3), and S4 (serotype 4), specifically stain the relevant homologous serotype without exhibiting any cross-reactivity against the other serotypes. Furthermore, the test is sensitive enough to differentiate at least twofold (0.3 log) differences in virus titer. The results of testing four individual experimental vaccine lots three or more consecutive times showed that all four lots contained similar proportions of the four vaccine strains as detected by the classical plaque neutralization identification test. The rapidity and efficiency of the FFIDA are desirable attributes that make it suitable for use in studies requiring identification and quantitation of one or more of the four major rotavirus serotypes.     

Direct detection of Streptococcus mutans in human dental plaque by polymerase chain reaction

Streptococcus mutans is an etiological agent in human dental caries. A method for the detection of S. mutans directly from human dental plaque by polymerase chain reaction has been developed. Oligonucleotide primers specific for a portion of the dextranase gene (dexA) of S. mutans Ingbritt (serotype c) were designed to amplify a 1272-bp DNA fragment by polymerase chain reaction. The present method specifically detected S. mutans (serotypes c, e and f), but none of the other mutans streptococci: S. cricetus (serotype a), S. rattus (serotype b), S. sobrinus (serotypes d and g), and S. downei (serotype h), other gram-positive bacteria (16 strains of 12 species of cocci and 18 strains of 12 species of bacilli) nor gram-negative bacteria (1 strain of 1 species of cocci and 20 strains of 18 species of bacilli). The method was capable of detecting 1 pg of the chromosomal DNA purified from S. mutans Ingbritt and as few as 12 colony-forming units of S. mutans cells. The S. mutans cells in human dental plaque were also directly detected. Seventy clinical isolates of S. mutans isolated from the dental plaque of 8 patients were all positive by the polymerase chain reaction. These results suggest that the dexA polymerase chain reaction is suitable for the specific detection and identification of S. mutans.     

Genetic and antigenic analysis of human rotavirus prevalent in Al-Taif, Saudi Arabia

The subgroup, serotype and electropherotype diversity of human rotavirus strains was investigated in Al-Taif, Saudi Arabia. Out of 349 faecal samples collected from diarrhoeic children, 150 (43 percent) tested rotavirus positive by a group-A specific enzyme-linked immunosorbent assay (ELISA). The majority (87 percent) of the infected children were below 2 years of age. Subgrouping and serotyping of rotaviruses with specific monoclonal antibodies showed that of the 150 rotavirus positive specimens, 17 percent belonged to subgroup I, 59 per cent belonged to subgroup II, and 24 percent were neither subgroup I nor subgroup II. The specimens were typed, as serotype 1 (43 percent), serotype 2 (5 percent), serotype 3 (11 percent), serotype 4 (10 percent) or mixed serotypes (3 percent). The remaining 41 (27 percent) specimens were untypeable. None of the serotypes showed association with a particular age group. An electrophoretic analysis of viral RNA revealed 11 distinct patterns (six long and five short). The majority, 78 percent were long patterns and 22 percent were short patterns. Analysis of the specimens for which subgroups, serotypes and electropherotypes were available indicated that a given RNA pattern does not correspond to a particular subgroup or serotype.     

An automated fluorescent PCR method for detection of shiga toxin-producing Escherichia coli in foods

An automated fluorescence-based PCR system (a model AG-9600 AmpliSensor analyzer) was investigated to determine whether it could detect Shiga toxin-producing Escherichia coli (STEC). The AmpliSensor PCR assay involves amplification-mediated disruption of a fluorogenic DNA signal duplex (AmpliSensor) that is homologous to conserved target sequences in a 323-bp amplified fragment of Shiga toxin genes stx1, stx2, and stxe. Using the Amplisensor assay, we detected 113 strains of STEC belonging to 50 different serotypes, while 18 strains of non-Shiga-toxin-producing E. coli and 68 strains of other bacteria were not detected. The detection limits of the assay were less than 1 to 5 CFU per PCR mixture when pure cultures of five reference strains were used and 3 CFU per 25 g of food when spiked ground beef samples that were preenriched overnight were used. The performance of the assay was also evaluated by using 53 naturally contaminated meat samples and 48 raw milk samples. Thirty-two STEC-positive samples that were confirmed to be positive by the culture assay were found to be positive when the AmpliSensor assay was used. Nine samples that were found to be positive when the PCR assay was used were culture negative. The system described here is an automated PCR-based system that can be used for detection of all serotypes of STEC in food or clinical samples.     

Management of chronic pain among elderly patients

OBJECTIVE: To identify the Staphylococcus aureus capsular serotypes that are not typable, using capsular serotypes 5 and 8, which are currently used to type S aureus isolated from cows with mastitis. SAMPLE POPULATION: Milk samples (n = 273) from cows with mastitis in 178 dairy herds in California, Wisconsin, Michigan, Texas, and New York that were collected by state diagnostic laboratories and S aureus-positive milk samples collected by Veterinary Health Services in the United Kingdom (15), France (22), The Netherlands (36), and Germany (21). PROCEDURE: Capsular serotyping of coded isolates was performed by use of direct cell agglutination and immunoprecipitation of cell extracts with antisera specific for capsular types 5 and 8 and a newly developed S aureus serotyping antiserum 336. RESULTS: In the United States, S aureus capsular types 5 and 8 accounted for 18 and 23% of the isolates, respectively, and type 336 accounted for 59%. Percentage of capsular serotypes in European samples were as follows: type 5 = 34%, type 8 = 34%, type 336 = 30%, and nontypable = 2%. CONCLUSIONS: Serotypes 5 and 8 accounted for only 41% of S aureus isolates from US milk samples, but accounted for 70% of isolates from European milk samples. Addition of the newly developed serotyping antiserum 336 to the typing scheme accounted for 100% of US samples and 98% of European samples and will enable development of a more comprehensive S aureus vaccine.     

Isolation of a specific chromosomic DNA sequence of Bacillus anthracis and its possible use in diagnosis

A 277-bp long DNA fragment, Ba813, was isolated from an avirulent Bacillus anthracis strain 7700 genomic library. Two oligonucleotides derived from the Ba813 sequence were used as primers in polymerase chain reaction tests on genomic DNA from 28 Bacillus anthracis and from 33 heterologous bacteria strains. A specific, 152-bp long DNA fragment was amplified only when Bacillus anthracis DNA was used as the target. The amplified product was analysed by non-radioactive sandwich hybridisation in microtiter plates using two oligonucleotides. The capture oligonucleotide C1 was covalently linked onto aminated wells of microtiter plates. The detection oligonucleotide D3 was labelled with biotine. The hybrid molecules were detected by avidine conjugated with alkaline phosphatase and chromogenic substrate. Amplification of Ba813 sequence may provide the basis for rapid and reliable assay for the detection and identification of Bacillus anthracis.     

Characterization of a monoclonal antibody against a nuclear antigen associated with serotype-1 Marek's disease virus-infected and transformed cells

A monoclonal antibody (MAb), 2BN90, was characterized on a panel of Marek's disease virus (MDV) isolates representing three antigenically related serotypes and lymphoblastoid cell lines established from Marek's disease (MD) tumors. It was reactive with all serotype 1 isolates, including CV1988, in both enzyme-linked immunosorbent assay (ELISA) and immunofluorescence (IF) test. The MAb immunoprecipitated and immunoblotted a 40-kilodalton protein, which was found to be phosphorylated by metabolic labeling with 32P inorganic phosphate, and localized to the cell nucleus using the IF test. It reacted with MDV-transformed lymphoblastoid cell lines, and the percentage of reactive cells was enhanced after treatment with iododeoxyuridine. MAb 2BN90 may be useful as a type-specific antibody of choice for serotyping MDV serotype 1 strains and as a probe for studying the role of pp40 in MDV infection and transformation.