Mycoplasma Strains Research Articles

Mycoplasmas in the etiology of multifactorial respiratory disease

The avian mycoplasmas pathogenic for commercial poultry, Mycoplasma gallisepticum and Mycoplasma synoviae in chickens and turkeys, and Mycoplasma meleagridis and Mycoplasma iowae in turkeys are egg-transmitted infections and exhibit wide variations in clinical manifestations. Mycoplasma gallisepticum strains vary widely in virulence, tissue tropism, and antigenic makeup and have the ability to alter the expression of major surface antigenic proteins. Although less well studied, strains of M. synoviae, M. meleagridis, and M. iowae appear to exhibit similar variability. Intraspecies variability among mycoplasma strains and their ability to interact with other disease-producing factors explain the wide variability of clinical manifestations, difficulties in diagnosis, their ability to persist within the host for long periods of time, and many of the difficulties involved in control and eradication programs. Mycoplasmas are also well known for their interactions with other infectious agents and environmental factors in producing clinical disease. Control of the clinical manifestations of Mycoplasma infections is simplified when concurrent infections are minimized and optimum environmental conditions are provided.

Genetic and serological analysis of the immunogenic 67-kDa lipoprotein of Mycoplasma sp. bovine group 7

The gene encoding a lipoprotein of 67 kDa, named P67, was cloned from Mycoplasma sp. bovine group 7 strain PG50 and expressed in Escherichia coli K12. Analysis of the amino acid sequence derived from the DNA sequence of the P67 gene revealed a typical prokaryotic signal peptidase II membrane lipoprotein lipid attachment site and a transmembrane structure domain in the leader sequence at the amino-terminal end of the protein. Protein P67 showed 91% identical amino acid residues to the lipoprotein P72 of Mycoplasma mycoides subsp. mycoides small colony type (SC) and 53% identical amino acid residues to a peptide of an unassigned gene on the genome of Mycoplasma capricolum subsp. capricolum. Antibodies made against recombinant P67 reacted with a 67-kDa protein in all Mycoplasma sp. bovine group 7 strains tested and also, to some extent, with P72 of Mycoplasma mycoides subsp. mycoides SC. The gene encoding P67 was present in all strains of Mycoplasma sp. bovine group 7 analysed, but not in other Mycoplasma sp. of the “mycoides cluster” and not in the phylogenetically related Mycoplasma putrefaciens. PCR and restriction fragment analysis revealed that the gene of P67 is conserved in all strains of Mycoplasma sp. bovine group 7. A specific PCR reaction based on the P67 gene sequence enabled rapid identification of strains belonging to Mycoplasma sp. bovine group 7.

Quality-control testing of mycoplasma medium.

Mycoplasmas lack many of the biosynthetic pathways of higher organisms; consequently, their growth and survival depend on an external supply of a wide variety of nutrients. Because they are very fastidious in their nutritional requirements, a complex growth medium is required for their culture. Although the exact requirements of most mycoplasma species are still incompletely defined, most can be grown in the laboratory in both liquid and solid agar media formulated to comprise as a minimum: a broth base, yeast extract, serum source, and solutions of pure chemicals. Details of components added to any particular medium will vary, according to the species of mycoplasma it is intended to culture (see Chapters 4– 6). It is clear that such a large number of components must inevitably introduce a degree of variability to the growth-promoting properties of the mycoplasma media between formulation lots. Consequently, it is of paramount importance that the introduction of any new component into a formulation and the preparation of media lots should always be accompanied by a quality validation with mycoplasma strains that the particular formulation is intended to support. This quality-control (QC) testing should cover all components, including preparations of pure chemicals (e.g., glucose and arginine) because although intrinsically these chemicals are unlikely to vary between lots supplied by the same manufacturer in the same way that broth base, serum, and yeast extract might, it is still necessary to exclude the possibility that errors may have occurred during their preparation (see Note 1).

Effect of Drinking Water Containing Ammonium Chloride or Sodium Bicarbonate on Mycoplasma gallisepticum Isolation in Experimentally Infected Broiler Chickens

In each of three trials, 150 day-old broiler chicks were eyedrop inoculated with 0.04 ml of high-passage F strain Mycoplasma gallisepticum (MG) and housed in biological isolation units at 10 chicks per unit. At 4 wk of age, 50 chickens were designated as controls and remained on tap water (pH 7.30), 50 chickens were provided tap water containing 0.63% ammonium chloride (NH4Cl, pH 6.91), and 50 chickens were provided tap water containing 1.26% sodium bicarbonate (NaHCO3, pH 8.17). Fluids were supplied for ad libitum consumption. At 5 wk of age, all chickens were swabbed from the choanal cleft for MG isolation and bled from the left cutanea ulnea vein for pH determination. As a percent of total swabs obtained, significantly fewer chickens that consumed water containing NH4Cl (38.3%) were positive for MG by culture compared with either the NaHCO3 group (61.3%) or the control group (67.6%). Nonmycoplasmal swab contamination was significantly higher for chickens that consumed water containing NH4Cl (59.7%) compared with either controls (31.8%) or NaHCO3-treated chickens (38.7%). When contaminated cultures were discarded, MG isolations from the tap water group were not significantly different from MG isolations from either the NH4Cl or NaHCO3 group. However, MG isolations from the NH4Cl group (95%) were significantly less compared with the NaHCO3 group (100%). Mortality was significantly higher in chickens that consumed water containing NaHCO3 (8.7%) compared with either controls (1.3%) or the NH4Cl-treated chickens (0.7%). Blood pH values were lower for the NH4Cl group (7.927), higher for the NaHCO3 group (8.093), and intermediate for controls (8.035). Results of this study suggest that water containing NH4Cl hinders the bacteriological recovery of MG from the choanal cleft.

The Effects of F Strain Mycoplasma gallisepticum, Mycoplasma synoviae, and the Dual Infection in Commercial Layer Hens over a 44-Week Laying Cycle When Challenged before Beginning of Lay. I. Egg Production and Selected Egg Quality Parameters

In each of two trials, 160 commercial pullets were separated into four treatments with four replicates of 10 chickens in each treatment. Forty pullets were designated as controls and received no inoculation; 40 other pullets received F strain Mycoplasma gallisepticum (FMG); an additional 40 pullets received Mycoplasma synoviae (MS); and the final 40 pullets were inoculated with both FMG and MS (dual). Hen-day egg production, egg weight, eggshell strength, Haugh unit score, pimpling incidence, and blood/meat spot incidence were monitored and recorded in each trial through an entire laying cycle. No significant difference was observed among the treatments for hen-day egg production, egg weight, eggshell strength, or Haugh unit scores. Significant differences were observed for pimpling incidence among controls (1.63%), Mycoplasma gallisepticum (MG)-infected (2.09%), and dual-infected hens (2.41%). A significant difference in blood/meat spot incidence was observed between MG-infected hens (0.27%) and dual-infected hens (0.45%). Histopathologic examination of the ovary and all segments of the oviduct revealed no significant differences among the treatments. These results suggest that the majority of the hen reproductive tract functions similarly in FMG-vaccinated, MS-infected, or dual-infected hens as compared with Mycoplasma-clean hens.

Comparative susceptibilities of various animal-pathogenic mycoplasmas to fluoroquinolones.

The in vitro activities of six antimicrobial agents were tested against 162 mycoplasma strains of eight species isolated from poultry and livestock at different geographic sites. Tiamulin was most active (MICs at which 90% of the isolates were inhibited [MIC90s], 0.025 to 0.25 microg/ml); enrofloxacin and danofloxacin had near equivalent activities (MIC90s, 0.05 to 1.0 microg/ml), but were much more active than flumequine (MIC90s, 1 to 50 microg/ml). The MIC90s of tylosin and oxytetracycline were 0.25 to > 100 microg/ml and 0.25 to 100 microg/ml, respectively.

Mycoplasma crocodyli sp. nov., a new species from crocodiles.

Organisms with the typical characteristics of mycoplasmas were isolated from joints and lungs of crocodiles. The results of growth inhibition tests and immunobinding assays showed that the 24 mycoplasma strains isolated were identical and distinct from previously described Mycoplasma, Entomoplasma, Mesoplasma, and Acholeplasma species. These organisms represent a new species, for which the name Mycoplasma crocodyli is proposed. M. crocodyli ferments glucose and maltose, does not produce films and spots, does not hydrolyze arginine, esculin, and urea, reduces tetrazolium chloride, and possesses phosphatase activity. It lyses and adsorbs bovine, ovine, and rabbit erythrocytes. Cholesterol or serum is required for growth. The optimum growth temperature is 37 degrees C. The G + C content of the DNA is 27.6 mol%. This organism causes exudative polyarthritis in crocodiles. The type strain of M. crocodyli is strain MP145 (= ATCC 51981).

Epitope Diversity of F Strain Mycoplasma gallisepticum Detected by Flow Cytometry

A culture of F strain Mycoplasma gallisepticum (F-MG) that exhibited an epitope identified by monoclonal antibody (MAb) 6F10 was used to inoculate leghorn hens in two different trials. In Trial 1, mature hens chronically infected with F-MG were swabbed at intervals from 230 to 345 days postinoculation (PI). The F-MG isolates were tested with an agar plate fluorescent antibody (APFA) method that used a polyclonal antibody and with a flow cytometry (PC) technique that used MAb 6F10. Primary cultures of swabs taken at 258, 272, 293, 318, and 345 days PI were all identified as positive by APFA, whereas FC identified 23%-41% as positive. Subsequently, MAb 6B11 was found, which reacted positively with isolates negative to MAb 6F10. Both 6F10 and 6B11 were used in the second trial, which was designed to identify F-MG isolates that were negative to 6F10. In Trial 2, naive birds were inoculated with F-MG when they were 9 wk old and were sampled at six intervals from 13 to 154 days PI. The APFA method was used to identify primary isolation (P0) cultures, and FC was performed on P0 cultures and the same cultures after they had been passed three times (third serial passage [P3] cultures). The APFA test identified 100% of the P0 cultures as F-MG. The FC results on P0 cultures showed 34.5% as 6F10 positive and 85.1% as 6B11 positive. Results for FC on P3 cultures showed 92.3% 6F10 positive and 96.3% 6B11 positive. These results suggest that the microenvironment of the colonization site in the hen induced an epitope diversity in F-MG, as evidenced by the loss in the expression of MAb 6F10-defined epitope. Isolation of the organism from hens and propagation for several in vitro passages resulted in the re-expression of the epitope defined by MAb 6F10.

The development of lameness and bone deformity in the broiler following experimental infection with Mycoplasma gallisepticum or Mycoplasma synoviae

Summary Groups of eight 7‐day‐old Mycoplasma‐free broiler chicks were inoculated with one of two strains of M. gallisepticum (MG/S6, MG/B31/85) or one of two strains of M. synoviae (MS/B31/88 and MS/B94/91) into the left hock joint. Controls were similarly inoculated with sterile mycoplasma broth. The birds were assessed at 2, 3, 4 and 5 weeks of age (1, 2, 3 and 4 weeks post‐injection) for lameness and joint and limb lesions. At 5 weeks of age they were killed and examined for gross skeletal abnormalities and microscopic lesions of both hock joints. The in‐contact controls showed no joint swelling or lameness. The mycoplasma did not apparently spread to cause gross pathology in the joints of in‐contact control birds, or to other joints in a bird injected with mycoplasma. Each mycoplasma strain caused specific pathologies manifest as varying degrees of lameness, left hock joint swelling, bone deformity and differences in the severity of articular cartilage and chondrodystrophic growth plate pathology. Shortening and bowing of the right tibiotarsus in the birds injected with mycoplasma indicated unilateral increased limb loading contributing to bone deformity. Chondrodystrophy seen with three mycoplasmal strains may have contributed to bone deformity in these birds. Lameness appeared to be a consequence of either joint swelling and/or bone deformity resulting in mechanical dysfunction. The role of joint pain in the lameness was not determined.

A 13-kDa Protein with a Helix–Turn–Helix Motif Is Encoded by Bacterial Operons Related to the SRP Pathway

We have identified a 13 kDa protein (p13) inMycoplasma mycoidessubsp.mycoidesthat is encoded immediately downstream of a protein homologous toE. coliFtsY, a protein taking part in the bacterial signal recognition particle (SRP) pathway. The same organisation of the p13 and FtsY genes occurs inMycoplasma pneumoniae.PCR analysis of different mycoplasma strains revealed the same organisation in strains belonging to theMycoplasma mycoidescluster of the mycoplasma phylogenetic tree. Searches in sequence databases identified homologues to p13 inBacillus subtilisandStreptococcus mutans.In these bacteria the p13 protein is encoded by the same operon as a protein homologous to the 54 kDa subunit of SRP. These findings suggest that there is a functional relationship between the p13 protein and the SRP pathway. Sequence analysis of the p13 proteins strongly suggest that they have a helix–turn–helix (HTH) motif, indicating that they are gene regulatory proteins.

Identification of two pathogenic avian mycoplasmas as strains of Mycoplasma pullorum.

Two mycoplasma strains were isolated from dead turkey embryos. Growth properties, biochemical and serological characteristics, and protein profiles indicated that these strains were closely related to Mycoplasma pullorum CKKT (T = type strain). This was confirmed by 16S rRNA sequence analysis, and the phylogenetic position of M. pullorum was established. Pathogenicity studies showed that the two strains, as well as M. pullorum CKKT, induced a statistically significant level of mortality after inoculation into chicken embryos.

Identification of Mycoplasma Isolates by ELISA

An antigen-based enzyme-linked immunosorbent assay (ELISA) was developed for the rapid identification of Mycoplasma gallisepticum and Mycoplasma synoviae. Two trials were conducted to compare the ELISA with the conventional fluorescent antibody technique. In trial 1, broilers were inoculated with M. gallisepticum F, M. gallisepticum S6, and M. synoviae. In trial 2, hens were inoculated with M. gallisepticum F, M. synoviae, and a combination of the two. Swab isolates were obtained from the choanal cleft and cultures were grown in broth media with a pH indicator. The organisms were bound to 96-well plates for the ELISA or cultured on agar for the fluorescent antibody test. Monoclonal antibodies directed to M. gallisepticum F, M. gallisepticum S6, and M. synoviae were reacted with the ELISA aliquots. The ELISA test identified the mycoplasmas in these samples and was completed within 8 hr but did not identify as many isolates as the fluorescent antibody procedure. The principal advantage of this ELISA is the ability to identify the strain of mycoplasma.

Morphological evidence of a filamentous cilia-associated respiratory (CAR) bacillus in goats.

A filamentous cilia-associated respiratory (CAR) bacillus was discovered in 12 3-4 month-old goats experimentally infected with two different strains of mycoplasmas belonging to the Mycoplasma mycoides type. The CAR bacilli were always arranged parallel to the cilia, and the morphology of these bacilli is very similar to that of other bacilli described previously in other species from various parts of the world.

Epidemiological study of Mycoplasma pneumoniae infections in japan based on PCR-restriction fragment length polymorphism of the P1 cytadhesin gene.

Two hundred fifty strains of Mycoplasma pneumoniae isolated during the past 20 years in Japan were classified into two groups (I and II) based upon different PCR-restriction fragment length polymorphism patterns of their P1 cytadhesin genes. Clear shifts between the M. pneumoniae groups were observed but did not appear to be correlated with M. pneumoniae epidemic cycles. Patients' sera showed relatively higher levels of antiadhesin antibodies to M. pneumoniae strains homologous with the infecting strain.

Variant colony surface antigenic phenotypes within mycoplasma strain populations: implications for species identification and strain standardization.

Immunobinding assays with mycoplasma colonies on agar plates (immunofluorescence and immunoperoxidase techniques) or with imprints of colonies transferred to solid supports (colony immunoblotting) are widely used as standard diagnostic tests for serological species identification of mycoplasma isolates. However, in light of the high rate of variability of surface antigens in many mycoplasmas, diagnostic data obtained with these techniques require a more critical evaluation. In this report, we demonstrate with some examples that mycoplasma surface variability based on alterations in expression, in size, and in surface presentation of integral and peripheral membrane proteins may lead to misinterpretation of colony immunostaining reactions obtained by using specific monoclonal antibodies as well as conventional diagnostic hyperimmune sera. To more easily identify phenotypically mixed isolates or samples which contain more than one species, we have introduced some minor modifications of the colony immunoblot technique which provide sharp signals of positive as well as negative reactions and enable identification of cryptic epitopes. It is further demonstrated that because of the variability in colony surface antigenic phenotype, mycoplasma strains, including well-established reference and other prototype strains which are used under the same designation in many laboratories, can differ markedly in their antigen profiles and their potentially virulence-related surface properties, since they are usually purified by filter cloning and often propagated by subcultivation of randomly selected agar-grown subpopulations. We conclude from this study that because of this surface variability, the establishment of criteria for standardization of mycoplasma strains and diagnostic antisera is urgently required in order to obtain reproducible results in different laboratories.

Genetic properties of Mycoplasma mycoides subspecies mycoides LC strains isolated from a goat in Japan in 1991.

Field isolated Mycoplasma strains LG1 and SF1 were isolated from the lung and leg joint fluid of a goat, respectively in Japan in 1991. These strains were closely related and had the following biochemical properties: glucose was metabolized, arginine and urea were not hydrolyzed, tetrazolium was reduced, phosphatase produced, and casein and coagulated serum were digested. Serologically, these strains cross-reacted with three Mycoplasma species [M. mycoides subsp. mycoides (Mmm), M. mycoides subsp. capri, and M. sp. Group 7]. From the above results, the field isolates were considered to be Mmm. In addition, DNA-DNA relatedness between the field isolates and other Mycoplasma species called the "mycoides cluster" was determined, and a recently developed PCR method was also used for mycoplasmal identification. From these results, the field isolates were identified as Mmm LC type.

A Mycoplasma strain F38 growth-inhibiting monoclonal antibody (WM-25) identifies an epitope on a surface-exposed polysaccharide antigen.

Monoclonal antibody (MAb) WM-25 differentiates by in vitro growth inhibition Mycoplasma capricolum subsp. capripneumoniae (Mycoplasma strain F38), which causes contagious carpine pleuropneumonia, from other Mycoplasma spp. (F. R. Rurangirwa, T. C. McGuire, A. J. Musoke, and A. Kibor, Infect. Immun. 55:3219-3220, 1987). The antigen identified by MAb WM-25 was isolated from solubilized Mycoplasma strain F38 organisms by MAb WM-25 affinity chromatography and was stained with Schiff's reagent, but not with Coomassie blue, after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment of purified F38 polysaccharide with periodate abolished binding with MAb WM-25, and MAb WM-25 binding was blocked with laminarin, a complex oligosaccharide with beta(1-->3) sugar linkages. Purified F38 polysaccharide blocked both growth inhibition and agglutination of live F38 organisms caused by MAb WM-25 and rabbit antiserum to F38 organisms. The results in this paper demonstrate that MAb WM-25 binds a periodate-sensitive epitope on the F38 polysaccharide which is also exposed on the surface of Mycoplasma strain F38. Because MAb WM-25 also causes in vitro growth inhibition of F38, the reactive polysaccharide epitope may induce protective immune responses.

In-vitro antimycoplasmal activity of flurithromycin

The in vitro activity of flurithromycin, a 14-membered macrolide drug, was found to be similar to that of erythromycin against 41 strains of Mycoplasma spp. and 100 strains of Ureaplasma urealyticum. All 28 strains of Mycoplasma hominis were uniformly resistant to both macrolides with MICs > 256 mg/L, U. urealyticum showed intermediate resistance with MIC50s of 0.5 and 1 mg/L for erythromycin and flurithromycin, respectively, whereas the ten strains of Mycoplasma pneumonia were susceptible to < or = 0.03 mg/L of both macrolides.

Phylogenetic analysis of mycoplasma strain ISM1499 and its assignment to the Acholeplasma oculi strain cluster.

A mycoplasma strain designated ISM1499 was used to develop a mycoplasma genetic system (G. G. Mahairas and F. C. Minion, J. Bacteriol. 171:1775-1780, 1989; G. G. Mahairas, C. Jian, and F. C. Minion, Gene 93:61-65, 1990), but phenotypic inconsistencies led to the conclusion that this organism had been classified incorrectly as a member of the species Mycoplasma pulmonis. Studies were initiated to determine the proper taxonomic position of ISM1499, and on the basis of the results of our genetic analysis, this strain was assigned to the Acholeplasma oculi strain cluster. The base composition of strain ISM1499 was identical to the base composition of A. oculi 19L, but not to the base composition of Acholeplasma laidlawii PG8 (28.3 and 30.7 mol% G+C, respectively). The taxonomic position of ISM1499 was examined by performing a parsimony analysis with 16S ribosomal DNA sequence data, and the results were compared with previous phylogenetic reconstructions. Our results indicated that ISM1499 is more closely related phylogenetically to A. oculi 19L than to A. laidlawii PG8 and JA1. Heterogeneity in the 16S ribosomal DNA sequences of A. oculi 19L and ISM1499 and in the 16S ribosomal DNA sequences of A. laidlawii PG8 and JA1 may indicate that unusual dissimilarities occur in the 16S ribosomal DNA sequences of members of the genus Acholeplasma.

Chronic Endometritis and Positive Mycoplasma Cultures: Is There a Correlation?

Objective: This study was undertaken to assess the impact of mycoplasma strains (Mycoplasma hominis or Ureaplasma urealyticum) on the development of chronic endometritis. Methods: Fifty-eight patients with acute pelvic infection were enrolled in this prospective cohort study. Endometrial cultures and biopsies were obtained on admission and 5–7 and 21–28 days after completion of treatment. Results: Of 148 samples, 40 were positive for mycoplasma strains (group A) and 58 were positive for mycoplasma with other pathogens (group B). Twenty-seven samples were positive for other pathogens only (group C). Chronic endometritis was seen in 7 (17.5%), 30 (51.7%), and 10 (37%) in group A, B, and C patients, respectively. Conclusions: The presence of mycoplasma strains in the endometrial cavity was not found to be associated with an increased incidence of chronic endometritis.