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 ~  Results
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Year : 2004  |  Volume : 22  |  Issue : 3  |  Page : 169-171
 

Relative susceptibility of six continuous cell lines for cultivation of chlamydia trachomatis strains


L & T Microbiology Research Center, Vision Research Foundation, Sankara Nethralaya, Chennai - 600 006, Tamil Nadu, India

Date of Submission22-Aug-2003
Date of Acceptance03-Oct-2003

Correspondence Address:
L & T Microbiology Research Center, Vision Research Foundation, Sankara Nethralaya, Chennai - 600 006, Tamil Nadu, India

 ~ Abstract 

Since susceptibility of a cell line is an important factor for cultivation of Chlamydia trachomatis, McCoy, HeLa, BHK-21, HEp-2, Vero and A549 cell lines were tested for this characteristic. These were inoculated with 150 infection-forming units (IFU) of C. trachomatis A, B, Ba and C serovars. Growth was graded according to the number of IFUs per microscopic field (100X). A549-cell line was not susceptible to infection by any of the serovars. The growth of C. trachomatis was good to very good in McCoy and HeLa cell lines. Vero, BHK-21 and HEp-2 cell lines varied considerably in the susceptibility to infection.

How to cite this article:
Malathi J, Shyamala G, Madhavan H N. Relative susceptibility of six continuous cell lines for cultivation of chlamydia trachomatis strains. Indian J Med Microbiol 2004;22:169-71


How to cite this URL:
Malathi J, Shyamala G, Madhavan H N. Relative susceptibility of six continuous cell lines for cultivation of chlamydia trachomatis strains. Indian J Med Microbiol [serial online] 2004 [cited 2019 Jun 19];22:169-71. Available from: http://www.ijmm.org/text.asp?2004/22/3/169/11211


McCoy is the most common cell line used for isolation and maintenance of C. trachomatis in the laboratory, but cell lines like HeLa, HEp-2, BHK-21 and Vero have also been used. For the past 12 years, our laboratory has been using stationary (cycloheximide-treated) McCoy cell line for cultivation of C. trachomatis from clinical specimens and for research with instances of failure of its growth from clinical samples that were smear and polymerase chain reaction positive. Though antibiotic treatment of the patient prior to collection of clinical specimen was suggested as the reason, susceptibility of the cell lines could also be an important factor. Therefore, this study was undertaken to compare the susceptibility of the commonly available continuous cell lines to the infectivity of C. trachomatis.

 ~ Materials and Methods Top

C. trachomatis strains
The standard ATCC strains of C. trachomatis serovar A (ATCC VR571 B), serovar B (ATCC VR-573), serovar Ba (ATCC VR-347) and serovar C (ATCC VR-572) obtained from Centers for disease control, Atlanta, Georgia, USA and maintained in McCoy cell line, were used in this study. Each stock strain was diluted in Eagle's maintenance medium (MM) without fetal calf serum and calibrated as to contain 150 elementary bodies (EBs) or infection-forming units (IFU) per 100無. This was done by counting the number of IFUs in all microscopic fields under 100X magnification (20X objective and 5X eyepiece) in the smear (prepared by using 1 無 of the stock suspension of the strain) stained by immunofluorescence (IF) using monoclonal antibodies raised against the major outer membrane protein (MOMP) of C. trachomatis (Orion diagnostics, Finland). Fluorescence microscope with a blue filter (Optiphot, Nikon, Japan) was used for scanning the IF stained smears.
Cell lines and growth susceptibility experiments
McCoy, HeLa, BHK-21, HEp-2, Vero and A549 cell lines obtained from National Center for Cell Science, Pune and maintained in our tissue culture laboratory, were used. They were subcultured over sterile cover slips placed inside glass shell vials as described by us earlier for McCoy cell line.[1] Cells grown over the cover slips were pretreated with maintenance medium (MM) containing cycloheximide (1痢/mL) at 370C overnight followed by inoculation with 100無 containing approximately 150 IFUs of each of the four standard strains. The shell vials were centrifuged at 3,000 rpm for 1 hour and 900 無 of MM containing cycloheximide (1痢/mL) and 10% fetal calf serum, L-glutamine (200mM) prepared in Eagle's minimum essential medium (Eagle's MEM) was added. Cultures were incubated at 370C for 48 hours, MM was aspirated and the cover slips were fixed with methanol. These cover slips were stained by IF for MOMP antigen of C. trachomatis. The experiments were simultaneously performed with all the cell lines with the same aliquots of the stock of each strain and MM. The IF stained cover slip preparations were observed under fluorescence microscope. The growth of each serovar in each of the cell line was graded according to the average number of IFUs per microscopic field under 100X magnification counted in 100 fields [Figure - 1]. The results were recorded as follows: average IFU counts of 50-65 as ++++ (very good) or more, 35-49 as +++ (good), 20-34 as ++ (moderate) and 1-19 as + (scanty).

 ~ Results Top

The [Table - 1] shows the results of the susceptibilities of the six cell lines to infection of the four strains of C. trachomatis. A549 cell line was not susceptible to infection by all the serovars of C. trachomatis. Other cell lines were susceptible to its infection in varying degrees. Serovar A showed very good growth in McCoy; HeLa and BHK-21 cell lines with moderate growth in HEp-2 and Vero cell lines. Growth of serovar B was good in McCoy and HeLa cell lines, moderate in BHK-21 and Vero cell lines and scanty growth in HEp-2 cell line. Serovar Ba showed good growth in McCoy and HeLa cell lines with moderate growth in HEp-2 cell line and scanty growth in Vero and BHK-21 cell lines. C. trachomatis serovar C showed very good to good growth in all the cell lines with no growth in A549 cell line.

 ~ Discussion Top

A uniform inoculum amount (150 IFU/100無) of C. trachomatis was used through out the study as the bacterium could be diluted to undetectable level by FAT unless multiplication of the bacterium occurred. The grading of the growth of C. trachomatis was done after due consideration of this characteristic of the bacterium experienced in our laboratory during the last 12 years.
A549 cell line was resistant to infection by C. trachomatis and may not be of use for its isolation. McCoy and HeLa were determined to be the most susceptible cell lines with very good growth of all four strains in them. Though serovar C was a chick embryo yolk sac adapted strain, its growth was very good in McCoy, HeLa, BHK-21 and Vero cell lines with good growth in HEp-2. In our study Vero had shown variable results with the four strains tested. Though HEp-2 cell line has been utilized for cultivation of C. trachomatis our study indicated that this cell line may not be useful for this purpose.[2],[3]
This study clearly showed that apart from the most commonly used McCoy cell line for the growth of C. trachomatis, HeLa and to some extent BHK-21 and Vero cell lines could also be used. Use of at least two of them for a clinical specimen may be helpful. 

 ~ References Top

1.Madhavan HN. Detection of Chlamydia trachomatis antigen in McCoy cells inoculated with conjunctival specimens by modified shell vial method using immunoperoxidase stain. Afro-Asian J Ophthalmol 1992;41:117-119.  Back to cited text no. 1    
2.Freise J, Gerard HC, Bunke T, Whittum-Hudson JA, Zeidler H, Kohler L, Hudson AP, Kuipers JG. Optimised sample DNA preparation for detection of Chlamydia trachomatis in synovial tissue by polymerase chain reaction and ligase chain reaction. Ann Rheum Dis 2001;60:140-145.  Back to cited text no. 2    
3.Kuipers JG, Scharmann K, Wollenhaupt J, Nettelnbreker E, Hopf S, Zeidler H. Sensitivities of PCR, Microtrak, Chlamydia EIA, IDEIA and PACE 2 for purified Chlamydia trachomatis elementary bodies in urine, peripheral blood, peripheral blood leukocytes and synovial fluid. J Clin Microbiol 1995;33:3186-3190.  Back to cited text no. 3    
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2004 - Indian Journal of Medical Microbiology
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