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Biomedical subjects

K Sachse

Publications and source records attributed to K Sachse.

At least 19 recordsLinked to original sources
PubMed2000

Comparison of the 16S-23S rRNA intergenic spacer regions among strains of the Mycoplasma mycoides cluster, and reassessment of the taxonomic position of Mycoplasma sp. bovine group 7.

Nucleotide sequence analysis of the 16S-23S rRNA intergenic spacer regions of six type or reference strains belonging to the Mycoplasma mycoides cluster and of Mycoplasma putrefaciens suggested the presence of two subclusters. One subcluster comprised M. mycoides subsp. mycoides small colony (SC) type, M. mycoides subsp. mycoides large colony (LC) type and M. mycoides subsp. capri, whereas the second subcluster comprised Mycoplasma capricolum subsp. capricolum, M. capricolum subsp. capripneumoniae and Mycoplasma sp. bovine group 7. The type strains from M. mycoides subsp. mycoides SC and M. mycoides subsp. capri had identical spacer sequences. The existence of two subclusters was supported by predicted secondary structures of the analysed region. The nucleotide variations in the loop domains of the secondary structures might be a useful genetic marker to distinguish between the two subclusters. The secondary structure differences delineated the differences between the two subclusters more clearly than the nucleotide sequence alignments, which only showed a small number of differences, and some of these were common to both clusters. The data also provided evidence in favour of a reclassification of Mycoplasma sp. bovine group 7 as another subspecies of M. capricolum.

Animals↗
PubMed2000

Epitope mapping of immunogenic and adhesive structures in repetitive domains of Mycoplasma bovis variable surface lipoproteins.

The family of variable surface lipoproteins (Vsps) of the bovine pathogen Mycoplasma bovis includes some of the most immunogenic antigens of this microorganism. Vsps were shown to undergo high-frequency phase and size variations and to possess extensive reiterated coding sequences extending from the N-terminal end to the C-terminal end of the Vsp molecule. In the present study, mapping experiments were conducted to detect regions with immunogenicity and/or adhesion sites in repetitive domains of four Vsp antigens of M. bovis, VspA, VspB, VspE, and VspF. In enzyme-linked immunosorbent assay experiments, sera obtained from naturally infected cattle showed antibodies to different repeating peptide units of the Vsps, particularly to units R(A)1, R(A)2, R(A)4.1, R(B)2.1, R(E)1, and R(F)1, all of which were found to contain immunodominant epitopes of three to seven amino acids. Competitive adherence trials revealed that a number of oligopeptides derived from various repeating units of VspA, VspB, VspE, and VspF partially inhibited cytoadhesion of M. bovis PG45 to embryonic bovine lung cells. Consequently, putative adherence sites were identified in the same repeating units (R(A)1, R(A)2, R(A)4.1, R(B)2.1, R(E)1, and R(F)1) and in R(F)2. The positions and lengths of the antigenic determinants were mostly identical to those of adhesion-mediating sites in all short repeating units, whereas in the considerably longer R(F)1 unit (84 amino acid residues), there was only one case of identity among four immunogenic epitopes and six adherence sites. The identification of epitopes and adhesive structures in repetitive domains of Vsp molecules is consistent with the highly immunogenic nature observed for several members of the Vsp family and suggests a possible function for these Vsp molecules as complex adherence-mediating regions in pathogenesis.

Amino Acid Sequence↗
PubMed2000

[Demonstration of Chlamydia from an equine abortion].

The isolation and identification of a chlamydial agent from an equine fetus is reported. The fetus was aborted by a mare with respiratory disease and fever in the 9th month of pregnancy. The serum of the mare was investigated by the compliment fixation test. Specific antibodies were detected for chlamydial antigen in a titer of > 1:40 and for equine herpes virus 1 antigen in a titer of 1:32. Pathological lesions were not found in the organs of the fetus. Chlamydiae were detected in the placenta by ELISA and subsequently isolated by cell culture. Using PCR technique the agent was identified as Chlamydophila psittaci.

Abortion, Veterinary↗
PubMed1999

Enhancement of Mycoplasma bovis detection in milk samples by antigen capture prior to PCR.

A rapid and sensitive procedure to detect Mycoplasma bovis from milk samples was developed. Mycoplasma cells were collected by selective binding to a monoclonal antibody, thus separating them from milk proteins and facilitating DNA extraction. Polymerase chain reaction (PCR) using species-specific primers allowed detection of 20 cfu ml-1 in agarose gels and 2 cfu ml-1 on Southern blots. The method is especially suitable for specimens from subclinical cases and intermittent shedders.

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

Intraspecies polymorphism of vsp genes and expression profiles of variable surface protein antigens (Vsps) in field isolates of Mycoplasma bovis.

To assess the extent of interstrain variation, 50 isolates of Mycoplasma (M.) bovis including the type strain PG45 were examined for the presence of a family of variable membrane surface lipoproteins (Vsps) and their genes. Southern hybridization using a genomic fragment carrying three distinct vsp genes (vspAEF) revealed a striking heterogeneity, with only 2/50 strains having identical banding patterns. Cluster analysis of the data showed that most isolates from interrelated herds (groups 1, 2 and 3) were combined in a cluster of 50% homology, while isolates from distinct geographical regions (groups 4, 5 and 6) were linked only at 18% homology. Vsp antigen expression was monitored by Western immunoblotting using four specific monoclonal antibodies (MAbs). Resembling the findings at the DNA level, interstrain variation of Vsp expression among groups 1-3 was less pronounced than among non-interrelated isolates from groups 4-6. Ten out of 50 strains did not hybridize with the vspAEF gene probe at high-stringency conditions, 8/50 failed to react with any of the Vsp-related MAbs, and 6/50 proved negative in both assays. Interestingly, most of these isolates produced hybridization signals at low stringency suggesting major distinctions in their vsp gene structure. The extensive evidence obtained on interstrain vsp gene polymorphism and variation in Vsp expression could provide a basis for a future understanding of the pathogenic potential of individual M. bovis strains.

Animals↗
PubMed1996

A PCR scheme for differentiation of organisms belonging to the Mycoplasma mycoides cluster.

Rapid and specific detection of organisms belonging to the Mycoplasma mycoides cluster, among them Mycoplasma (M.) mycoides subsp. mycoides SC, the agent of contagious bovine pleuropneumonia, is an important condition for effective control of the respective animal diseases. In an effort to improve diagnosis, a PCR identification scheme for these mycoplasmas was developed. A set of primer combinations derived from the CAP-21 genomic regions of member organisms of the Mycoplasma mycoides cluster was selected by means of which complete differentiation within the cluster can be accomplished. Nested PCR involving cluster-specific amplification at the first stage and group-specific amplification using internal primers at the second stage was shown to be applicable for identification of all six groups forming the cluster. For example, external primers P1 /P2 and internal primers P6/P7 were used to distinguish M. mycoides subsp. mycoides SC from LC strains. Using the present PCR procedure, identification of mycoplasmas could be considerably accelerated in comparison to conventional methods (5 h vs. one week) and specificity was also improved.

Animals↗
PubMed1996

Mechanisms and factors involved in Mycoplasma bovis adhesion to host cells.

Mycoplasma (M.) bovis cytadhesion was studied using permanent embryonic bovine lung (EBL) cells as host system. Adherence rates were found to be strongly dependent on temperature and the mycoplasma-to-EBL ratio near the point of saturation of the attachment isotherm was determined to be 225 : 1. Mild trypsinization of viable M. bovis cells caused a measurable decrease of adherence indicating that surface proteins, among them the P26 antigen, played a major part as adhesion factors. Neuraminidase treatment of mycoplasmas led to a drastic reduction of adherence rates, which emphasizes the importance of sialyl moieties in adhesive interactions. The ability of the P26 antigen, a hydrophilic 32-kDa protein, to function as an adhesin was confirmed using a competitive adherence assay, in which the HPLC-purified protein was shown to reduce mycoplasma adhesion. These data complement previous findings obtained with the corresponding monoclonal antibody (MAb) 4F6. In further inhibition experiments, it could be demonstrated that MAb 1E5, which is directed against a common epitope of at least three members of the Vsp (variable surface protein) family of M. bovis, was also capable of decreasing mycoplasma attachment to EBL cells. This is the first evidence of possible involvement of Vsps in cytadhesion. In an effort to identify more putative adhesion proteins of this organism, the reverse adherence screening assay was used, a procedure based on the specific binding of labelled mammalian tissue culture cells to Western-blotted mycoplasmal proteins.

Adhesins, Bacterial↗
PubMed1996

Rapid detection of Mycoplasma bovis in milk samples and nasal swabs using the polymerase chain reaction.

Two methods are suggested that allow direct detection of Mycoplasma bovis from clinical samples, i.e. milk and nasal swabs, respectively. Milk samples were trypsinized in the presence of Triton X-100 and passed through a DNA-binding filter membrane, from which DNA was extracted and subjected to PCR. The detection limit was 500 cfu ml-1 on agarose gels and 50 cfu ml-1 after Southern hybridization so that the method can be used to monitor low-titre samples from animals at the subclinical stage. Results became available within 24 h, thus rendering the procedure more rapid than ELISA and culture techniques. Six other methods designed for milk or other complex samples were tested in this study, but found unsatisfactory. Rapid and specific detection of the pathogen by PCR from nasal mucus treated with lysis buffer and proteinase K was demonstrated for swabs taken from experimentally infected calves. Both methods represent useful tools for effective livestock monitoring and single-animal diagnosis.

Animals↗
PubMed1996

Mycoplasma bovis as an agent of mastitis, pneumonia, arthritis and genital disorders in cattle.

Bovine diseases due to Mycoplasma bovis can cause considerable economic losses in cattle production. While the pathogen is principally responsible for therapy-resistant mastitis on large dairy farms, on smaller farms the typical mycoplasma diseases are calf pneumonia and arthritis. Moreover, the pathogen is able to cause genital disorders. M. bovis infection can be controlled effectively only if appropriate measures are implemented at the earliest possible stage. Since immunoprophylaxis and antibiotic treatment are known to be ineffective, control measures must include the introduction of strict hygiene standards, the restriction of animal movement out of infected herds and the culling of clinically diseased animals and shedders of the mycoplasma (the latter only in the case of mastitis and genital disorders). In this review, symptoms of the various diseases caused by M. bovis are described and characteristics of the course of infection are outlined. To clarify the origin and spread of the infection, the authors describe the main properties and reservoirs of the pathogen and summarise experimental evidence on modes of transmission to susceptible organs. As effective diagnosis is a prerequisite for the introduction of early control measures, the advantages and disadvantages of currently used diagnostic methods are discussed in detail. It is a serious shortcoming if testing for mycoplasmas is not included in routine bacterial examination of clinical samples. As a consequence, some M. bovis infections will remain undetected and outbreaks cannot be controlled properly. Finally, practical recommendations are given for prevention and control, including the formation of mycoplasma-free herds.

Animals↗
PubMed1994

Antigen heterogeneity among isolates of Mycoplasma bovis is generated by high-frequency variation of diverse membrane surface proteins.

The protein and antigen profiles of 11 isolates of Mycoplasma bovis were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis of whole organisms. The isolates examined included the type strain PG45 and 10 other filter-cloned strains or purified isolates both from animals without clinical signs and from clinical cases of bovine mastitis, arthritis, or pneumonia. While the overall protein patterns visualized by silver staining were very similar, marked differences in the antigen banding profiles were detected by rabbit antiserum prepared against whole organisms from one of the strains analyzed. This antigenic heterogeneity was shown to be independent of the geographical origin, the type of clinical disease, and the site of isolation and was also observed among serial isolates from a single animal. Antigen profiles were further monitored throughout sequentially subcloned populations of the PG45 strain. This clonal analysis revealed a high-frequency variation in the expression levels of several prominent antigens. All of these variable antigens were defined by detergent-phase fractionation with Triton X-114 as amphiphilic integral membrane proteins. A subset of different-sized membrane proteins was identified by a monoclonal antibody raised against a PG45 subclone expressing a 63- and a 46-kDa variant antigen within that set. The selective susceptibility of these proteins to trypsin treatment of intact organisms and their ability to bind the monoclonal antibody in colony immunoblots demonstrated that they were exposed on the cell surface. In addition, their preferential recognition by serum antibodies from individual cattle with naturally induced M. bovis mastitis or arthritis confirmed that they were major immunogens of this organism. These studies establish that the apparent antigenic heterogeneity among M. bovis isolates reported here does not represent stable phenotypic strain differences generated from accumulated mutational events but reflects distinct expression patterns of diverse, highly variable membrane surface proteins.

Animals↗
PubMed1993

Inhibition of Mycoplasma bovis cytadherence by a monoclonal antibody and various carbohydrate substances.

The attachment of Mycoplasma bovis to permanent embryonic bovine lung (EBL) cells was studied in order to identify factors participating in the adhesion process. A monoclonal antibody directed against a 26 kDa protein of M. bovis was shown to reduce cytadherence of strains 120 and 454 by 46% and 70%, respectively. In uninhibited assays, strain 120 which exhibits an intense 26 kDa band in electrophoretic protein patterns adhered more strongly to EBL cell monolayers than strain 454 whose corresponding band is considerably weaker. The findings indicate involvement of the 26 kDa protein in M. bovis adherence. In further inhibition experiments, the ability of N-acetyl-neuraminlactose, glycophorin and dextran sulfate to significantly reduce adherence could be demonstrated. This suggests participation of sialic acid residues and probably also sulfatide groups as binding receptors. The data point to a complex adhesion mechanism with similarities to M. pneumoniae.

Animals↗
PubMed1993

Antigen capture ELISA using a monoclonal antibody for the detection of Mycoplasma bovis in milk.

An enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody (mAb) was developed to detect Mycoplasma (M.) bovis in milk samples from cattle. With this procedure, 1 x 10(5) colony forming units per ml (cfu/ml) milk were routinely detectable. No cross-reactions to other bovine mycoplasma species were observed. Both the sensitivity of 80.6% and the specificity of 94.9% are sufficient for its use in diagnosis of clinical mastitis. The sensitivity could be increased by 10% after introduction of 48-hour pre-incubation of samples. This allowed recognition of cows shedding M. bovis amounts of 10(3) cfu/ml in their milk, which is typical for subclinical cases. Screening of milk samples by means of this antigen capture ELISA has advantages over culture methods in terms of speed and potential to monitor large herds, thereby permitting early culling of infected animals to reduce transmission of the pathogen to non-infected animals.

Animals↗
PubMed1993

Classification of Pasteurella field strains isolated from farms in Germany using traditional methods and DNA-DNA hybridization.

410 Pasteurella (P.) field strains isolated from calves and piglets were classified according to Bisgaard et al. (1). 376 strains were assigned to P. multocida ssp. multocida, 34 of them were ornithine- and trehalose+, and 61 of them ornithine- and trehalose-. 4 strains belonged to P. multocida ssp. septica, 4 to P. multocida ssp. gallicida, 6 to P. avium biovar 2 and 20 to P. canis biovar 2. There was no difference in the prevalence of the species in calves and pigs. The fact that strains belonging to P. multocida ssp. septica were isolated only from calves and P. multocida ssp. multocida ornithine- and trehalose- were mostly isolated from piglets could indicate a certain host specificity of these isolates. In genotypic investigations 20 field isolates of P. multocida belonging to different Carter serotypes, as well as serologically negative strains were compared to reference strains in terms of deoxyribonucleic acid (DNA) relatedness. The data obtained by filter hybridization revealed a considerable degree of genotypic intraspecies heterogeneity within P. multocida. No correlations to the respective serotypic classification could be detected.

Animal Husbandry↗
PubMed1993

Detection of Mycoplasma bovis using in vitro deoxyribonucleic acid amplification.

Detection of Mycoplasma bovis by traditional culture methods is rather time-consuming and is often hampered by bacterial contamination. The development of a rapid and specific alternative is, therefore, an important prerequisite in improving the diagnosis of bovine diseases caused by this agent. The authors have successfully used nucleic acid probes containing genomic restriction fragments of M. bovis cloned into the plasmid vector pUC19 for species-specific detection by dot blot hybridisation of small quantities of M. bovis deoxyribonucleic acid (DNA). The problem of direct M. bovis detection from contaminated milk could not be solved using this procedure. Therefore, further research was conducted using in vitro DNA amplification by polymerase chain reaction (PCR). Species-specific nucleic acid probes were sequenced and suitable PCR primers selected. Using the PCR procedure, ten colony-forming units (CFU) were detected from broth cultures and, after DNA isolation, the equivalent of 1 CFU was detected. Direct detection of M. bovis from biological samples proved extremely difficult due to protein interference. It was shown, however, that direct PCR detection from milk is possible after effective protein removal by combined extraction and protease digestion.

Animals↗