Sreeja R Nair
Characterization Of Genomic And Plasmid DNA Of Chlamydia psittaci Isolates - Mannuthy Department of Microbiology, College of Veterinary and Animal Sciences 2001
Four isolates of Chlamydia psittaci (M-28, M-430, M-121, P-156)
obtained from ruminant abortion were subjected to restriction enzyme analysis
and plasmid profiling to assess molecular level differences/homogenity among
the isolates. The changes produced in developing chick embryo and the
cytopathic effect in Me Coy cell line by the isolates were also investigated in
this study.
The C. psittaci isolates preserved in the Department of Microbiology
were revived by passaging in embryonated chicken eggs through yolk sac route.
Six to seven day-old developing chick embryo were used for propagation of the
isolates. The isolates, M-28 and M-430 were found to produce death of the
embryos within 10 days. The isolates M-121 and P-156 were not able to cause
death within 10 days. The embryos appeared stunted with patchy haemorrhages
all over the body and hyperemic legs. The yolk sac was thin walled, with
deeply injected blood vessels and the yolk was thin and watery. M-28 and
M-430 produced more severe lesions of the inoculated embryos compared with
the other isolates.
The isolates were grown in Me Coy cell line. All isolates revealed
marked CPE, characteristic of C. psittaci, with rounding and clumping of cells
to form syncytia at 48 h PI and formation of intra cytoplasmic inclusion bodies
by 96 h PI. The isolates M-121 and P-156 took more time for the production of
CPE compared with that of M-28 and M-430. The isolates were confirmed as
C. psittaci by fluorescence antibody technique which revealed, apple green
fluorescing chlamydial inclusions in the infected cell line by 96 h PI.
The CE as well as cell line propagated isolates were used as a source of
elementary bodies for DNA extraction. The purified EBs were prepared by
urografin density gradient centrifugation. After obtaining sufficient amount of
EBs, it was subjected to DNA extraction. The isolates M-28, M-430, M-121
and P-156 had DNA concentrations of 1710 ug/ml, 1130 Jlgl Iml, 1545 ug/ml
and 1475 ug/ml respectively.
Restriction enzyme digestion of all the isolates were done separately
with Eeo RI, Hae III and Barn HI.
Eeo RI cleaved DNA of the isolates M-430 and P-156 into rune
fragments, M-28 into eight and M-121 into ten fragments respectively. The
molecular size of bands for all isolates had variation in heavier fragments.
Common bands were observed at five regions for all isolates.
Hae III cleaved the caprine isolates into eight fragments and the bovine
isolate M-121 into seven and P-156 into nine fragments. Variation could be
observed on the molecular size of the fragments.
Bam HI digested all isolates except M-121 into eight fragments. For
M-121, seven fragments were obtained. Common bands were noticed at 0.2
and 0.7 kbp region.
All enzymes were found to be useful in the differentiation of C. psittaci
isolates as these REs produced variation as well as similarity in the restriction
fragment sizes.
Plasmid profiling revealed the absence of plasmids ill all the four
isolates screened.
Thus, a combination of phenotypic and genotypic methods could be
used as epidemiological tools in the differentiation of C. psittaci strains of
mammalian origin.
636.089 6 / SRE/CH
Characterization Of Genomic And Plasmid DNA Of Chlamydia psittaci Isolates - Mannuthy Department of Microbiology, College of Veterinary and Animal Sciences 2001
Four isolates of Chlamydia psittaci (M-28, M-430, M-121, P-156)
obtained from ruminant abortion were subjected to restriction enzyme analysis
and plasmid profiling to assess molecular level differences/homogenity among
the isolates. The changes produced in developing chick embryo and the
cytopathic effect in Me Coy cell line by the isolates were also investigated in
this study.
The C. psittaci isolates preserved in the Department of Microbiology
were revived by passaging in embryonated chicken eggs through yolk sac route.
Six to seven day-old developing chick embryo were used for propagation of the
isolates. The isolates, M-28 and M-430 were found to produce death of the
embryos within 10 days. The isolates M-121 and P-156 were not able to cause
death within 10 days. The embryos appeared stunted with patchy haemorrhages
all over the body and hyperemic legs. The yolk sac was thin walled, with
deeply injected blood vessels and the yolk was thin and watery. M-28 and
M-430 produced more severe lesions of the inoculated embryos compared with
the other isolates.
The isolates were grown in Me Coy cell line. All isolates revealed
marked CPE, characteristic of C. psittaci, with rounding and clumping of cells
to form syncytia at 48 h PI and formation of intra cytoplasmic inclusion bodies
by 96 h PI. The isolates M-121 and P-156 took more time for the production of
CPE compared with that of M-28 and M-430. The isolates were confirmed as
C. psittaci by fluorescence antibody technique which revealed, apple green
fluorescing chlamydial inclusions in the infected cell line by 96 h PI.
The CE as well as cell line propagated isolates were used as a source of
elementary bodies for DNA extraction. The purified EBs were prepared by
urografin density gradient centrifugation. After obtaining sufficient amount of
EBs, it was subjected to DNA extraction. The isolates M-28, M-430, M-121
and P-156 had DNA concentrations of 1710 ug/ml, 1130 Jlgl Iml, 1545 ug/ml
and 1475 ug/ml respectively.
Restriction enzyme digestion of all the isolates were done separately
with Eeo RI, Hae III and Barn HI.
Eeo RI cleaved DNA of the isolates M-430 and P-156 into rune
fragments, M-28 into eight and M-121 into ten fragments respectively. The
molecular size of bands for all isolates had variation in heavier fragments.
Common bands were observed at five regions for all isolates.
Hae III cleaved the caprine isolates into eight fragments and the bovine
isolate M-121 into seven and P-156 into nine fragments. Variation could be
observed on the molecular size of the fragments.
Bam HI digested all isolates except M-121 into eight fragments. For
M-121, seven fragments were obtained. Common bands were noticed at 0.2
and 0.7 kbp region.
All enzymes were found to be useful in the differentiation of C. psittaci
isolates as these REs produced variation as well as similarity in the restriction
fragment sizes.
Plasmid profiling revealed the absence of plasmids ill all the four
isolates screened.
Thus, a combination of phenotypic and genotypic methods could be
used as epidemiological tools in the differentiation of C. psittaci strains of
mammalian origin.
636.089 6 / SRE/CH