Assessing the in vitro effectiveness of antimicrobials against Mycoplasma mycoides subsp. mycoides small-colony type to reduce contagious bovine pleuropneumonia infection.

In vitro minimum inhibitory concentrations were determined for 21 antimicrobials against 41 isolates of Mycoplasma mycoides subsp. mycoides small-colony type, the cause of contagious bovine pleuropneumonia. Of the antimicrobials used most widely in Africa, oxytetracycline and tilmicosin were effective, while the isolates were resistant to tylosin. These results provide a baseline for monitoring antimicrobial resistance.

Capsular polysaccharide conjugate vaccines against contagious bovine pleuropneumonia: Immune responses and protection in mice.

The immunogenicity of Mycoplasma mycoides subsp. mycoides small colony biotype (MmmSC) vaccines was investigated in BALB/c mice. Groups of mice were vaccinated with either (1) unconjugated capsular polysaccharide (CPS), (2) CPS covalently conjugated to ovalbumin via a carbodiimide reaction, (3) CPS non-covalently bound to latex microspheres, (4) CPS non-covalently complexed with rabbit anti-CPS IgG, and (5) whole inactivated, ultrasonically disrupted (WID) MmmSC. Only mice immunized with the CPS-ovalbumin conjugate exhibited a significant (P<0 small middle dot001) antibody response against CPS. Mice immunized with WID vaccine exhibited a high ELISA antibody titre against non-CPS (protein) antigens only. Mice given WID vaccine were immune against challenge with live MmmSC, and exhibited a significantly reduced degree of mycoplasmaemia (both in incidence and duration) as compared with non-vaccinated controls (P<0 small middle dot001). Mice immunized with the CPS-ovalbumin conjugate did not exhibit a reduction in mycoplasmaemia. The bactericidal activity of rabbit MmmSC-antiserum in an in-vitro growth inhibition test was related to the CPS antibody titre. This was not observed with antisera from the vaccinated mice. None of the mouse antisera exhibited growth inhibiting activity, irrespective of a high CPS or protein antibody titre (CPS-ovalbumin or WID vaccine groups, respectively). Thus, it would seem that protection against an MmmSC-induced mycoplasmaemia in the mouse is based upon cell-mediated rather than humoral immunity. The results suggest that conjugation to ovalbumin significantly increases the antibody response to CPS in the mouse; the lack of bactericidal activity of mouse anti-CPS as compared with rabbit anti-CPS in vitro suggests either that the titre of growth inhibiting antibodies is lower in the mouse or that the mechanism of growth inhibition differs between antibodies of the two species.

Effect of HEPES buffer systems upon the pH, growth and survival of Mycoplasma mycoides subsp. mycoides small colony (MmmSC) vaccine cultures.

The use of a buffer system based on N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), in conjunction with standard Gourlay's culture medium was investigated for the growth and maintenance of Mycoplasma mycoides subsp. mycoides SC vaccine strain T(1)44. When the initial pH of the culture medium was adjusted to 8.0, 0.075 M HEPES-NaOH was found to be sufficient to prevent the pH falling below 7.1 at any stage during the growth cycle, even in the presence of 0.5% glucose. Compared to growth in standard unbuffered Gourlay's medium, the final culture titre was found to be one log(10) higher, at 10(11) colour changing units (CCU) per ml, and considerably extended culture survival was observed at 37 degrees C. The titre remained above 10(10) CCU ml(-1) for 4 days, and above 10(8) CCU ml(-1) in excess of 1 month. After 4 month's storage at 37 degrees C the titre had fallen to 5x10(4) CCU ml(-1). In contrast, no viable bacteria could be detected in standard unbuffered medium 3 days after the onset of stationary phase, at which point the pH had dropped to 5.4. No significant difference in growth rate between the two media was observed. Adoption of a HEPES-NaOH buffer system by African vaccine manufacturers should require minimal changes to current formulations and procedures, and should enhance both the final titre and thermostability of freeze-dried and liquid broth vaccines against contagious bovine pleuropneumonia (CBPP).

Rapid detection of contagious caprine pleuropneumonia using a Mycoplasma capricolum subsp. capripneumoniae capsular polysaccharide-specific antigen detection latex agglutination test.

Latex microspheres (diameter, 8 microm) were coated with anti-Mycoplasma capricolum subsp. capripneumoniae polyclonal immunoglobulin G (IgG) antiserum (anti-F38 biotype). The coated microspheres, when used in a latex agglutination test (LAT), detected M. capricolum subsp. capripneumoniae antigen in the serum of goats with contagious caprine pleuropneumoniae (CCPP). Beads also agglutinated strongly in the presence of purified M. capricolum subsp. capripneumoniae capsular polysaccharide (CPS). Preabsorption of CPS-specific antibodies prior to coating of the beads removed agglutinating activity in the presence of M. capricolum subsp. capripneumoniae, strongly suggesting that CPS is the likely soluble antigen recognized by the test. In addition, the specificity of the LAT exactly mirrored that of an M. capricolum subsp. capripneumoniae CPS-specific monoclonal antibody (WM25): of the 8 other mycoplasma species tested, agglutination was observed only with bovine serogroup 7. The LAT detected all 11 strains of M. capricolum subsp. capripneumoniae examined in this study, with a sensitivity level of 2 ng of CPS, or the equivalent of 1.7 x 10(4) CFU, in a reaction volume of 0.03 ml of serum. With field sera from goats with CCPP, the results of the LAT exhibited a 67% correlation with the results of the currently used complement fixation test (CFT), with the main discrepancy in diagnosis resulting from the increased sensitivity of the LAT compared to that of CFT. This antigen-detection LAT should prove particularly useful in identifying animals in the earliest stages of CCPP and combines sensitivity and low cost with ease of application in the field, without the need for any specialist training or equipment.

Extended stability of restriction enzymes at ambient temperatures.

The stability of restriction enzymes as supplied by manufacturers without any modification has been examined. No reduction in activity was observed for three enzymes (HindIII, EcoRI and Tsp509I) held at ambient temperature or 4 degrees C for the period of study (12 months), while activity was observed for up to 12 weeks after storage at 37 degrees C, which was considerably better than following desiccation with trehalose, a recognized preservation technique. A larger trial of 23 different restriction enzymes held at room temperature for one week showed that all enzymes retained significant activity. As a practical demonstration of the usefulness of this finding, enzymes were posted to Africa by conventional mail (cost $1 US) and shown to retain activity upon arrival after three weeks in transit (compared to a cost of $1000 US by cold-chain transportation). Supplying enzymes to third-world markets should now be possible by removing the necessity for cold-chain transport. After arrival, enzymes can simply be stored in a standard domestic refrigerator.

Characterization of strains of Mycoplasma mycoides subsp. mycoides small colony type isolated from recent outbreaks of contagious bovine pleuropneumonia in Botswana and Tanzania: evidence for a new biotype.

Four strains of Mycoplasma mycoides subsp. mycoides small colony type (MmmSC) isolated from recent outbreaks of contagious bovine pleuropneumonia (CBPP) in Africa have been investigated. One Botswanan strain, M375, displayed numerous and significant phenotypic differences from both contemporary field isolates and older field and vaccine strains (African, Australian, and European strains dating back to 1936). Differences include altered morphology, reduced capsular polysaccharide production, high sensitivity to MmmSC rabbit hyperimmune antisera in vitro, and unique polymorphisms following immunoblotting. While insertion sequence analysis using IS1634 clearly indicates a close evolutionary relationship to west African strains, hybridization with IS1296 shows the absence of a band present in all other strains of MmmSC examined. The data suggest that a deletion has occurred in strain M375, which may explain its altered phenotype, including poor growth in vitro and a relative inability to cause septicemia in mice. These characteristics are also exhibited by Mycoplasma capricolum subsp. capripneumoniae (causal agent of contagious caprine pleuropneumonia [CCPP]), against which M375 antiserum exhibited some activity in vitro (unique among the various MmmSC antisera tested). These findings may have evolutionary implications, since CCPP is believed to be lung specific and without a septicemic phase (unlike CBPP). Since M375 was isolated from a clinical case of CBPP, this novel biotype may be fairly widespread but not normally isolated due to difficulty of culture and/or a potentially altered disease syndrome. Bovine convalescent antisera (obtained from contemporary naturally infected cattle in Botswana) were active against strain M375 in an in vitro growth inhibition test but not against any other strains of MmmSC tested. There exists the possibility therefore, that strain M375 may possess a set of protective antigens different from those of other strains of MmmSC (including vaccine strains). These findings have implications for the control of the current CBPP epidemic in Africa.

Effect of active immunization against recombinant-derived chicken prolactin fusion protein on the onset of broodiness and photoinduced egg laying in bantam hens.

The hypothesis that the onset of incubation behaviour (broodiness) in the domestic hen is induced by an increase in prolactin secretion was investigated by actively immunizing bantam hens against recombinant-derived chicken prolactin. A second objective was to establish whether active immunization against prolactin affects photoinduced onset of egg laying and the rate of egg production. The immunogen was a fusion protein (beta gals-prolactin, 23 kDa) produced in Escherichia coli, comprising chicken prolactin (without the nine amino-terminal amino acids) fused to 18 amino acids of E. coli beta-galactosidase. A control immunogen was produced in the same strain of E. coli harbouring the same plasmid vector used to produce beta gals-prolactin minus the prolactin gene sequence. Hens were immunized i.m. with 1 mg of protein containing 0.8-0.9 mg of fusion protein in Freund's incomplete adjuvant at 4-8 week intervals beginning before or after egg laying, which was induced by increasing the daily photoperiod. The beta gals-prolactin immunogen, but not the control immunogen, stimulated the production of antibodies to chicken prolactin. In Expts 1, 2 and 3, hens were placed in floor pens with nest boxes after photostimulation to induce broodiness. In these experiments, immunization with beta gals-prolactin reduced the incidence or delayed the development of broodiness. This effect was more pronounced if immunization was initiated before, rather than after, the onset of egg laying. In Expts 1 and 2 hens were immunized with beta gals-prolactin before photostimulation. The presence of antibodies to prolactin in their blood did not affect photoinduced onset of egg laying.(ABSTRACT TRUNCATED AT 250 WORDS)

A possible role for the pcnB gene product of Escherichia coli in modulating RNA: RNA interactions.

The sequence of the PcnB protein of Escherichia coli, a protein required for copy number maintenance of ColE1-related plasmids, was compared with the PIR sequence database. Strong local similarities to the sequence of the E. coli protein tRNA nucleotidyltransferase were found. Since a substrate of the latter protein, tRNA, structurally resembles the RNAs that control ColE1 copy number we believe that we may have identified a region in PcnB that interacts with these RNAs. Consistent with this idea is our observation that PcnB is required for the replication of R1, a plasmid whose replication is also regulated by a small RNA.

Cloning and characterization of an Escherichia coli gene, pcnB, affecting plasmid copy number.

A gene, pcnB, affecting the copy number of ColE1-related plasmids has been cloned and mapped to 3.6 min on the Escherichia coli chromosome between panD and fhu. The gene encodes a previously undescribed 48 kD protein. Several independently isolated mutants exhibiting the same phenotype, reduced copy number, have been shown to be pcnB-.

A DNA fragment containing the groE genes can suppress mutations in the Escherichia coli dnaA gene.

An 8.2 kb fragment of E. coli chromosomal DNA, when cloned in increased copy number, suppresses the dnaA46 mutation, and an abundant protein of about 68 kd (60 kd when measured by us), encoded by the fragment, is essential for the suppression (Takeda and Hirota 1982). Mapping experiments show that the fragment originates from the 94 min region of the chromosome. It encodes several proteins but only one abundant polypeptide of the correct size, the product of the groEL gene. Suppression by the fragment is allele specific; those mutations which map to the centre of the gene are suppressed. Other initiation mutants including dnaA203, dnaA204, dnaA508, dnaAam, dnaC, dnaP and dnaB252 are not suppressed. Most suppressed strains are cold-sensitive suggesting an interaction between the mutant proteins (or their genes) and the suppressing protein or proteins.