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S Levisohn

Publications and source records attributed to S Levisohn.

At least 19 recordsLinked to original sources
PubMed2001

Use of arbitrarily primed polymerase chain reaction to investigate Mycoplasma bovis outbreaks.

Colony lineages from three Mycoplasma bovis outbreaks representing different husbandry conditions in the United States were characterized with arbitrarily primed polymerase chain reaction (AP-PCR). Cases studied included a closed beef herd, a dairy calf ranch, and a feedlot. The DNA was obtained from colony lineages and used for AP-PCR with primers REP1R-I and REP2-I. Case A and C lineages were uniform by AP-PCR analysis. Lineages from case B showed heterogeneity with AP-PCR. Outbreaks A and C were therefore both infected by one source, while the ranch (case B) was infected by multiple calf shipments. The AP-PCR typing method provides genotypic epidemiological information to successfully characterize M. bovis from sequential sampling of outbreaks and different husbandry conditions.

Animal Husbandry↗
PubMed2001

Molecular variability of the adhesin-encoding gene pvpA among Mycoplasma gallisepticum strains and its application in diagnosis.

Mycoplasma gallisepticum is an important pathogen of chickens and turkeys that causes considerable economic losses to the poultry industry worldwide. The reemergence of M. gallisepticum outbreaks among poultry, the increased use of live M. gallisepticum vaccines, and the detection of M. gallisepticum in game and free-flying song birds has strengthened the need for molecular diagnostic and strain differentiation tests. Molecular techniques, including restriction fragment length polymorphism of genomic DNA (RFLP) and PCR-based random amplification of polymorphic DNA (RAPD), have already been utilized as powerful tools to detect intraspecies variation. However, certain intrinsic drawbacks constrain the application of these methods. The main goal of this study was to determine the feasibility of using an M. gallisepticum-specific gene encoding a phase-variable putative adhesin protein (PvpA) as the target for molecular typing. This was accomplished using a pvpA PCR-RFLP assay. Size variations among PCR products and nucleotide divergence of the C-terminus-encoding region of the pvpA gene were the basis for strain differentiation. This method can be used for rapid differentiation of vaccine strains from field isolates by amplification directly from clinical samples without the need for isolation by culture. Moreover, molecular epidemiology of M. gallisepticum outbreaks can be performed using RFLP and/or sequence analysis of the pvpA gene.

Adhesins, Bacterial↗
PubMed2000

A chromosomal region of Mycoplasma agalactiae containing vsp-related genes undergoes in vivo rearrangement in naturally infected animals.

A Mycoplasma agalactiae genomic fragment carrying four vsp-related genes (designated avg: agalactiae variable genes) was cloned, sequenced and compared to the vspA gene of Mycoplasma bovis. The following features were revealed: (i) the presence of a highly conserved vsp 5' upstream region; (ii) a highly homologous vsp N-terminal end encoding a putative lipoprotein signal sequence; (iii) sequence divergence of the rest of the mature proteins. By using avg specific probes in Southern blot analysis of genomic DNAs of M. agalactiae strains as well as of isolates from infected animals, marked DNA polymorphism of avg fragments was demonstrated. In addition, the avg genomic fingerprints were monitored for a period of 7 months, in isolates of M. agalactiae from an individual chronically infected animal. The results provided evidence that a chromosomal region of M. agalactiae, carrying vsp-related genes, undergoes rearrangements in vivo in the natural animal host during the course of infection.

Animals↗
PubMed2000

Molecular characterization of the Mycoplasma gallisepticum pvpA gene which encodes a putative variable cytadhesin protein.

A putative cytadhesin-related protein (PvpA) undergoing variation in its expression was identified in the avian pathogen Mycoplasma gallisepticum. The pvpA gene was cloned, expressed in Escherichia coli, and sequenced. It exhibits 54 and 52% homology with the P30 and P32 cytadhesin proteins of the human pathogens Mycoplasma pneumoniae and Mycoplasma genitalium, respectively. In addition, 50% homology was found with the MGC2 cytadhesin of M. gallisepticum and 49% homology was found with a stretch of 205 amino acids of the cytadherence accessory protein HMW3 of M. pneumoniae. The PvpA molecule possesses a proline-rich carboxy-terminal region (28%) containing two identical directly repeated sequences of 52 amino acids and a tetrapeptide motif (Pro-Arg-Pro-X) which is repeated 14 times. Genetic analysis of several clonal isolates representing different expression states of the PvpA product ruled out chromosomal rearrangement as the mechanism for PvpA phase variation. The molecular basis of PvpA variation was revealed in a short tract of repeated GAA codons, encoding five successive glutamate resides, located in the N-terminal region and subject to frequent mutation generating an in-frame UAA stop codon. Size variation of the PvpA protein was observed among M. gallisepticum strains, ranging from 48 to 55 kDa and caused by several types of deletions occurring at the PvpA C-terminal end and within the two directly repeated sequences. By immunoelectron microscopy, the PvpA protein was localized on the mycoplasma cell surface, in particular on the terminal tip structure. Collectively, these findings suggest that PvpA is a newly identified variable surface cytadhesin protein of M. gallisepticum.

Adhesins, Bacterial↗
PubMed2000

Avian mycoplasmosis (Mycoplasma gallisepticum).

Mycoplasma gallisepticum is the most economically significant mycoplasma pathogen of poultry, and has a world-wide distribution. In common with other mycoplasmas, M. gallisepticum is minute in size with minimal genetic information and with a total lack of a bacterial cell wall. These properties are reflected in a high degree of interdependence between M. gallisepticum and the host animal, and in the fastidious nature of the organism in vitro. Strains of M. gallisepticum differ markedly with respect to important biological properties such as pathogenicity, infectivity, tissue tropism and transmissibility. In addition, phenotypic variation of major surface antigens occurs at high frequency, which is a probable explanation for chronic infection by M. gallisepticum despite a strong immune response. Infection with M. gallisepticum has a wide variety of clinical manifestations, but even in the absence of overt clinical signs, the economic impact may be significant. The most dramatic disease presentation of M. gallisepticum is chronic respiratory disease in meat-type birds, often as one of several aetiological agents in a multi-factorial disease complex. Transmission of M. gallisepticum in ovo from infected breeder birds to progeny is the major route of dissemination of the infection, and is the prime consideration for international trade. In most countries, control programmes for M. gallisepticum are based on maintaining commercial breeding stock free of infection. In instances where control of M. gallisepticum infection is not feasible, vaccination, especially with newly developed live M. gallisepticum vaccines, is being evaluated as an option. Major advances in diagnostic methods have been made in recent years. Control programmes have been based on serological methods, with screening for infection usually accomplished by the slide plate agglutination (SPA) test or by enzyme-linked immunosorbent assay. Further serological testing and/or demonstration of the presence of the organism must be used to confirm SPA suspected positive tests. In principle, detection of the presence of the M. gallisepticum organism can be by isolation of the organism or detection of the deoxyribonucleic acid by molecular methods. Polymerase chain reaction represents a rapid and sensitive alternative to traditional culture methods, which require time-consuming specialised techniques. The development of molecular typing methods affords new opportunities for epidemiological studies and identification of reservoirs of infection.

Animals↗
PubMed1999

The vsp locus of Mycoplasma bovis: gene organization and structural features.

Major lipoprotein antigens, known as variable membrane surface lipoproteins (Vsps), on the surface of the bovine pathogen Mycoplasma bovis were shown to spontaneously undergo noncoordinate phase variation between ON and OFF expression states. The high rate of Vsp phenotypic switching was also shown to be linked with DNA rearrangements that occur at high frequency in the M. bovis chromosome (I. Lysnyansky, R. Rosengarten, and D. Yogev, J. Bacteriol. 178:5395-5401, 1996). In the present study, 13 single-copy vsp genes organized in a chromosomal cluster were identified and characterized. All vsp genes encode highly conserved N-terminal domains for membrane insertion and lipoprotein processing but divergent mature Vsp proteins. About 80% of each vsp coding region is composed of reiterated coding sequences that create a periodic polypeptide structure. Eighteen distinct repetitive domains of different lengths and amino acid sequences are distributed within the products of the various vsp genes that are subject to size variation due to spontaneous insertions or deletions of these periodic units. Some of these repeats were found to be present in only one Vsp family member, whereas other repeats recurred at variable locations in several Vsps. Each vsp gene is also 5' linked to a highly homologous upstream region composed of two internal cassettes. The findings that rearrangement events are associated with Vsp phenotypic switching and that multiple regions of high sequence similarity are present upstream of the vsp genes and within the vsp coding regions suggest that modulation of the Vsp antigenic repertoire is determined by recombination processes that occur at a high frequency within the vsp locus of M. bovis.

5' Untranslated Regions↗
PubMed1998

Mycoplasma sturni from blue jays and northern mockingbirds with conjunctivitis in Florida.

Northern mockingbirds (Mimus polyglottos) and blue jays (Cyanocitta cristata) in a Florida (USA) wildlife care facility developed clinical signs and gross lesions suggestive of the ongoing outbreak of Mycoplasma gallisepticum (MG) conjunctivitis in house finches (Carpodacus mexicanus) and American goldfinches (Carduelis tristis). Mycoplasmal organisms were cultured from conjunctival/corneal swabs of birds with sinusitis, conjunctivitis, and/or epiphora. All of the isolates tested were identified as Mycoplasma sturni by indirect immunofluorescence. Mycoplasma sturni as well as MG should be considered in the differential diagnosis of songbirds with conjunctivitis.

Animals↗
PubMed1997

Genetic variation among Mycoplasma agalactiae isolates detected by the variant surface lipoprotein gene (vspA) of Mycoplasma bovis.

Multiple restriction fragments, homologous to the previously described Mycoplasma bovis vspA gene, were identified in the chromosome of Mycoplasma agalactiae. The vspA, a representative variable surface lipoprotein gene of the vsp gene family, and four synthetic oligonucleotides, representing sequences complementary to selected regions of the vsp genes, were used as probes against digested chromosomal DNAs of several M. agalactiae clinical isolates. The resulting Southern blot analysis demonstrated a marked DNA polymorphism of multiple vspA-related fragments among the isolates. An oligonucleotide representing a conserved 5'-region common to all known vsp genes, was found to hybridize to multiple M. agalactiae genomic fragments while the other three oligonucleotides, representing distinct repetitive structures within the coding region of three known vsp genes (vspA, vspE, and vspF), failed to react. These results argue for the possible existence of a gene family in M. agalactiae analogous to the vsp system of M. bovis but comprised of diverse genes.

Antigens, Bacterial↗
PubMed1997

Adherence of Mycoplasma gallisepticum involves variable surface membrane proteins.

Adherence of Mycoplasma gallisepticum to erythrocytes was examined by colony immunoblotting, detergent phase fractionation, trypsin treatment, comparison of protein profiles, and comparison of erythrocyte-bound mycoplasma protein fractions of hemadsorption-positive and -negative mutants. The binding of M. gallisepticum to chicken or human erythrocytes was found to be mediated via surface-exposed membrane proteins undergoing high-frequency phase variation.

Animals↗
PubMed1996

Use of species-specific oligonucleotide probes to detect Mycoplasma gallisepticum, M. synoviae, and M. iowae PCR amplification products.

Three digoxigenin-labeled oligonucleotide probes, complementary to the variable region of the 16S ribosomal RNA (rRNA) gene of Mycoplasma gallisepticum, M. synoviae, and M. iowae were designed. The oligonucleotides were used in a dot blot hybridization assay. The target DNA is a 780-bp fragment of the 16S rRNA gene of avian mycoplasmas amplified by a single set of primers (multispecies polymerase chain reaction [PCR]). The oligonucleotide probes were specific for their corresponding PCR products at hybridization conditions of 56 C and 50% formamide. The detection limit of the dot blot hybridization assay was approximately 70, 50, and 30 colony-forming units for M. gallisepticum, M. synoviae, and M. iowae, respectively, per 4 microliters of PCR. In general, the oligonucleotide probe dot blotting assay was a more sensitive and effective method of detecting PCR products than detection by gel electrophoresis.

Animals↗
PubMed1995

Cytokines induced in vitro by Mycoplasma mycoides ssp. mycoides, large colony type.

Septicemic disease in goats and sheep caused by the large colony type of Mycoplasma mycoides ssp. mycoides has clinical and pathological features in common with septic endotoxemia. We studied the ability of the mycoplasma to induce mediators of biological responses to endotoxin, such as TNF alpha, IL-1 alpha, IL-6 and nitric oxide. Heat-killed suspensions of mycoplasmas in a concentration of 25 micrograms protein ml-1 induced all mediators in macrophages from peritoneal cavity and bone marrow of both C3H/HeN (LPS responsive) and C3H/HeJ (LPS low-responsive) mice. Lipopolysaccharide (LPS) in a concentration of 100 ng ml-1 induced the mediators in C3H/HeN derived macrophages, only. Simultaneous stimulation of macrophages with interferon-gamma enhanced the secretion of nitric oxide (measured as nitrite) but not the cytokines. We conclude that heat-killed Mycoplasma mycoides ssp. mycoides, large colony type, has cytokine inductive activity similar to bacterial endotoxin, but with an induction mechanism different from LPS.

Animals↗
PubMed1995

In vivo variation of Mycoplasma gallisepticum antigen expression in experimentally infected chickens.

The antigen expression profiles of Mycoplasma gallisepticum isolates obtained from tracheal swabs of chickens after aerosol-inoculation with M. gallisepticum strain R or clonal variant R/E were examined in western immunoblots. A reference anti-M. gallisepticum chicken antiserum and antisera from individual infected chickens as well as monoclonal antibodies (mAbs) specific for surface proteins were used to monitor in vivo antigenic variation. mAbs 1E5 and 12D8, recognizing PvpA and p67a, recently shown to undergo high-frequency in vitro phase variation, were used for consecutive staining of colony and western immunoblots in order to distinguish between the resultant phenotypes with respect to the corresponding epitopes. Marked differences in the expression of major immunogenic proteins, including p67a, were observed between the two inocula as well as among reisolates recovered at different times of infection. Comparative western immunoblot analysis of the rapidly changing chicken serum antibody response and reisolates recovered during the course of an experimental infection with M. gallisepticum R or clonal variant R/E suggest that immune modulation may have a key role in generating surface diversity. In addition, comparison of colony immunoblots of strain R inoculum and of reisolated colonies from tracheas of birds 8 days post infection indicated an in vivo selection of the PvpA+p67a- phenotype. This study established that surface antigens of M. gallisepticum are subjected in vivo to rapid alteration in their expression. This variability may function as a crucial adaptive mechanism, enabling the organism to escape from the host immune defense and to adapt to the changing host environment at different stages of a natural infection.

Animals↗
PubMed1994

A surface epitope undergoing high-frequency phase variation is shared by Mycoplasma gallisepticum and Mycoplasma bovis.

We have recently reported that three distinct size- and phase-variable surface lipoproteins (Vsps) of the bovine pathogen Mycoplasma bovis possess a common epitope recognized by monoclonal antibody 1E5. In the present study, we show that this epitope is also present on a size-variant protein (PvpA) of the avian pathogen Mycoplasma gallisepticum. Application of monoclonal antibody 1E5 in Western immunoblot analysis of Triton X-114 phase-fractionated proteins and in colony immunoblots, as well as in trypsin and carboxypeptidase digestion experiments, has demonstrated that (i) PvpA is an integral membrane protein with a free C terminus, (ii) the shared epitope is surface exposed, and (iii) PvpA is subjected to high-frequency phase variation in expression. By using serum antibodies from M. gallisepticum-infected chickens, we were able to demonstrate the immunogenic nature of PvpA and identify three additional highly immunogenic Triton X-114 phase proteins (p67, p72, and p75) also undergoing high-frequency phase variation spontaneously and independently. Metabolic labeling experiments with [14C]palmitate and [14C]oleate revealed that PvpA, in contrast to p67, p72, and p75, is not lipid modified. Southern blot hybridization with restriction fragments carrying the pvpA gene of M. gallisepticum or the vspA gene of M. bovis against digested genomic DNA of the two Mycoplasma species indicated the absence of genetic relatedness between the pvpA and vspA genes. The apparent complexity of the antigenic variation phenomenon in M. gallisepticum is discussed.

Animals↗
PubMed1993

Species-specific oligonucleotide probes complementary to 16S rRNA of Mycoplasma gallisepticum and Mycoplasma synoviae.

Mycoplasma gallisepticum and M synoviae are important avian pathogens causing respiratory diseases which result in great economic losses in poultry farming. Two oligonucleotide probes, complementary to the variable region V8 of 16S rRNA from the avian mycoplasmas M gallisepticum and M synoviae have, therefore, been designed and used in direct filter hybridisation experiments. Both probes gave strong hybridisation signals with their homologous targets, whereas no cross-hybridisations were obtained with any of the other avian mycoplasmas tested. It was possible to detect 2-3 x 10(4) mycoplasma organisms by direct filter hybridisation experiments with radiolabelled probes. The probes were also used to analyse several laboratory strains and field isolates of M gallisepticum and M synoviae with complete agreement between the probe technique and the other methods used for species determination. Atypical M gallisepticum strains also gave strong hybridisation signals with the M gallisepticum specific probe.

Animals↗
PubMed1991

Use of an ELISA for differential diagnosis of Mycoplasma agalactiae and M mycoides subspecies mycoides (LC) in naturally infected goat herds.

Contagious agalactia is an ovine and caprine mycoplasmosis which manifests as mastitis, arthritis and keratoconjunctivitis. Mycoplasma agalactiae is recognised as a causal agent but M mycoides subspecies mycoides (LC), and M capricolum may also be responsible for this syndrome in goats. The clinical signs are not pathognomonic; diagnostic procedures are based on isolation of the organism from diseased animals or by detection of seroconversion. An ELISA specific for M agalactiae and M m mycoides (LC) is described. The specificity of the antigens was demonstrated by immunoblotting and by ELISA using monospecific hyperimmune rabbit sera. A correlation of ELISA activity with other serological tests and isolation of mycoplasmas was carried out in two goat herds under field conditions. Results indicate the ability to detect subclinical mycoplasma infection and individual carrier goats on the basis of ELISA, a finding which will assist control procedures.

Animals↗
PubMed1989

DNA probes for Mycoplasma gallisepticum and Mycoplasma synoviae: application in experimentally infected chickens.

DNA probes specific for Mycoplasma gallisepticum and M. synoviae were selected from genomic libraries prepared in the pUC13 vector. The probes hybridized with the DNA of a wide spectrum of strains within each homologous species, but did not react with the heterologous species or with DNA from any other avian mycoplasma or bacteria tested. Experimental infection and contact exposure of chickens to M. gallisepticum served as models to test the effectiveness of the DNA probe in diagnosis as compared with serological and culture detection methods carried out in parallel. A correlation was generally found between the level of M. gallisepticum in tracheal swabs and the effectiveness of the probe, although a predictably reactive level of mycoplasmas was not always detected. Treatment of clinical specimens with acetylcysteine to disrupt mucus improved the detection rate. Dot-blot hybridization with probe pMG4 enabled positive identification of M. gallisepticum at an early stage of infection, prior to the development of a serological response in the infected chicken. Results are obtainable within 4 days of sampling, much more rapidly than culture, and also in clinical specimens from which mycoplasma isolation is impossible, such as carcasses. The results indicate that the use of DNA probes for the early and rapid detection of M. gallisepticum infection is feasible; a development which can replace laborious culture techniques and less effective serological methods, and thus reduce the time required for diagnosis.

Animals↗
PubMed1989

Genetic and antigenic relatedness between Mycoplasma gallisepticum and Mycoplasma synoviae.

The two avian pathogens Mycoplasma gallisepticum and Mycoplasma synoviae were found, by Southern blot hybridization of their digested DNAs, to share genomic nucleotide sequences additional to those of the highly conserved ribosomal RNA genes. The assumption that some of the shared sequences encode for antigens or epitopes common to both mycoplasmas was supported by Western immunoblot analysis of cell proteins of one mycoplasma with specific antiserum to the other mycoplasma. Interestingly, the band patterns of reactive antigens were different for some of the M. gallisepticum strains, supporting the concept that the species is genotypically variable. The results of the present study may explain the cross-reactivity of the two mycoplasmas noted previously in a variety of routine serological tests.

Animals↗