Mycoplasma Iowae Species-Specific DNA Probe

Abstract

Abstract.A Mycoplasma iowae (MI) species-specific DNA probe (designated pMI-2) of 6.0 kbp (kilobase pairs) was isolated from an MI strain I-695 genomic library prepared in plasmid pUC8 and Escherichia coli strain JM83. When labeled with [32] P by nick translation, the probe hybridized in dot blot assays with 6 reference strains and 8 field isolates of MI but not with 16 other known species of avian mycoplasmas. The pMI-2 probe detected a minimum of 1.5 ng of MI strain I-695 chromosomal DNA. Under identical conditions of hybridization, the probe did not hybridize with a high concentration (200 ng) of M. gallisepticum or M. synoviae chromosomal DNA.Field and laboratory studies indicate that Mycoplasma iowae (MI) causes mortality of turkey embryos and airsacculitis, stunting, leg deformities, and exudative tenosynovitis in juvenile turkeys. [1][2][3]10,14 Strains of MI are homogeneous in some characteristics and heterogeneous in others. Thus, biochemical tests, immunofluorescence, and protein profiling by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) have tended to be group specific, whereas growth inhibition and restriction enzyme analysis of DNA patterns have tended to be strain-specific tests. 15The objective of this study was to develop a speciesspecific DNA probe that was sensitive and specific for the identification and differentiation of MI from other avian mycoplasmas. Materials and methodsMycoplasma cultures. Reference strains, a field isolates of MI, and type strains of 3 pathogenic and 13 nonpathogenic avian mycoplasms were included in the study. All species except M. synoviae (MS) were grown in PPLO broth base b with 12% horse serum and 10% fresh yeast extract. The MS was grown in Mycoplasma broth base, c containing 12% swine serum, 0.0125% NAD, and 0.0125% cysteine hydrochloride. 13DNA extraction and purification. The chromosomal DNA of MI strain I-695, M. gallisepticum (MG) strain S6, and MS strain WVU-1353 were isolated using published procedures. The isolated DNA was purified by cesium chloride gradient centrifugation. The concentration of the DNA was determined by fluorometry. Received for publication May 14, 1990. 25-µ1 reaction volume containing 1 × ligase buffer. After 3 cycles of incubation on ice for 5 min and at room temperature for 5 min, the reaction mixture was placed at 4 C overnight and then terminated with 1 µ1 of 0.5 M ethylenediaminetetraacetic acid (EDTA). Preparation of competent cells of Escherichia coli strain JM83. The described procedure8 for the preparation of competent cells was followed.Transformation. Twenty-five microliters of ligation solution, which contained 60 ng of MI DNA and 120 ng of plasmid pUC8, was added to 200 µ1 of competent E. coli cells, incubated on ice for 20 min, heat shocked at 42 C for 2 min, and immediately placed on ice for 5 min and then placed at room temperature for 5 min. Aliquots of 225 µ1 of transformed cells were plated on LB agar 9 containing 100 µg/ ml of ampicillin, 25 µg/ml isopropyl beta-D-thiogalacto-pyranoside (I...

Mycoplasma iowae in turkeys (Meleagris gallopavo)

“…There are early descriptions of the experimental use of PCR for detecting MI (Zhao and Yamamoto, 1990;Kempf et al, 1994;Boyle et al, 1995). It is a rapid, sensitive, and simple diagnostic method with Garcia et al (1996) describing the successful development and experimental use of PCR as part of a multi-mycoplasma species test that included MI.…”

“…This became known as the Iowa 695 strain of avian mycoplasma. It can also be used to identify some MI strains (Zhao and Yamamoto, 1990;Boyle et al, 1995;Cai et al, 2008). This has mostly been alleviated with the use of polymerase chain reaction (PCR) assays that have allowed the detection of species specific DNA segments.…”

Genome Sequence of Mycoplasma iowae Strain 695, an Unusual Pathogen Causing Deaths in Turkeys

“…ield and laboratory studies have identified that Mycoplasma iowae is the etiological agent of reduced hatchability, malabsorption syndrome, mild airsacculitis, leg deformities, and exudative tenosynovitis in turkeys and chickens (15). M. iowae strains isolated from turkeys, chickens, and wild birds were reported in North America, Western Europe, Israel, India, and Pakistan, with an unusual case isolated from aborted apple seeds in France in 1979 (2).…”