BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, researchlibraries, and research funders in the common goal of maximizing access to critical research.

Chemotaxis in Mycoplasma gallisepticumAuthor(s): K. M. LamSource: Avian Diseases, 49(1):152-154. 2005.Published By: American Association of Avian PathologistsDOI: http://dx.doi.org/10.1637/7232-070604RURL: http://www.bioone.org/doi/full/10.1637/7232-070604R

BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, andenvironmental sciences. BioOne provides a sustainable online platform for over 170 journals and books publishedby nonprofit societies, associations, museums, institutions, and presses.

Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance ofBioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use.

Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiriesor rights and permissions requests should be directed to the individual publisher as copyright holder.

Research Note—

Chemotaxis in Mycoplasma gallisepticum

K. M. Lam

Department of Population Health and Reproduction, School of Veterinary Medicine,University of California, Davis, CA 95616

Received 6 July 2004; Accepted 30 September 2004

SUMMARY. Boyden-type chemotactic chambers were used to demonstrate that Mycoplasma gallisepticum (MG) was capable ofmigrating into chemotactic membranes. Scanning electron microscopy was used to confirm that MG could penetrate themembranes. To further demonstrate the invasive ability of MG, MG was deposited on the shell membranes of 9-day-old chickenembryos, and after 6 days of incubation, the presence of MG DNA in the allantoic fluids was detected by polymerase chainreactions. These results indicate that MG can penetrate cellular membrane, possibly by going through the porous cellular surface.

RESUMEN. Nota de Investigacion—Quimiotaxis en Micoplasma gallisepticum.Se utilizaron camaras quimiotaxicas de Borden con la finalidad de demostrar la capacidad del Micoplasma gallisepticum (MG) de

migrar a traves de membranas quimiotaxicas. Se uso microscopıa electronica de barrido para demostrar que el MG podıa penetrarlas membranas. Con la finalidad de demostrar plenamente la capacidad invasora del MG, este organismo se deposito sobre lasmembranas de la cascara de embriones de pollo de 9 dıas de edad. Despues de 6 dıas de incubacion, utilizando la prueba de lareaccion en cadena por la polimerasa, se detecto en el fluido alantoideo la presencia de DNA correspondiente a MG. Estosresultados indican que el MG puede penetrar membranas celulares, posiblemente a traves de poros en la superficie celular.

Key words: Mycoplasma gallisepticum, chemotaxis, scanning electron microscopy

Abbreviations: FITC ¼ fluorescein isothiocyanate; fMLP ¼ N-formyl-methionyl-leucyl-phenylalanine; MG ¼ Mycoplasmagallisepticum; PBS ¼ phosphate-buffered saline; PCR ¼ polymerase chain reaction; SEM ¼ scanning electron microscopy

Mycoplasma gallisepticum (MG) is known to have a terminal blebstructure (12), which is used to attach intimately to the surface ofcells (7,9,10). MG is also known to be motile, moving in thedirection of the blebs, albeit very slowly (3). However, it is unknownwhether Mycoplasma can undergo chemotaxis, and there is still noincontrovertible evidence showing that MG can migrate from thesurface to the inner environment of cells, although confocalmicroscopy (2,13), electron microscopy (11), and immunohisto-chemistry (11) data have suggested that possibility. In this research,chemotactic membranes and eggshell membranes were used todemonstrate that MG could penetrate the membrane.

MATERIALS AND METHODS

MG. The S6 strain of MG was used in the following experiments.The organisms were propagated in B medium (4) for 48 hr, thenaliquotted in 1-ml amounts, and stored frozen at �80 C. The thawedMG culture had a titer of 13 104 colony-forming units per 0.1 ml.Chicken eggs. Fertile chicken eggs were purchased from a local

vendor and were incubated under standard conditions. The status ofMycoplasma infection or contamination in the purchased eggs wasunknown; however, periodic testing of the allantoic fluids of the eggs bypolymerase chain reaction (PCR) was negative for the presence of MG.On the ninth day of incubation, the eggs were candled and the nonfertileones were discarded. A hole was punched on the air-cell end of theembryo, and 100 ll of freshly thawed MG culture or B medium wasdeposited on the shell membranes of the eggs with a tuberculin syringe.Two such experiments were performed: the first one consisted of 60embryos and the second, 58 embryos, each time with two thirds of theembryos inoculated with MG and the other one third with B medium.After the hole was sealed with household glue, the embryos were returnedto the incubator. At 0, 1, 3, 5, and 7 days after MG inoculation, and inanother experiment at 0, 2, 4, and 6 days after inoculation, 4 embryoswere opened and their allantoic fluids were collected, aliquotted in 1.5-mlamounts, and stored frozen at�80 C until used.

Extraction of MG DNA and PCR. The frozen allantoic fluid wasthawed and centrifuged at 2000 rpm to precipitate the particulatematerials. The supernatant was transferred to a fresh tube andcentrifuged at 12,0003 g for 15 min to precipitate MG. After washingthe pellet once with phosphate-buffered saline (PBS), 1 ml of distilledwater was added to resuspend the pellet; one half of the resuspendedMG was boiled for 5 min to release its DNA, and the remaining half wastreated with lysis buffer and with phenol-chloroform to extract the DNAto be used in PCRs. The primers used in PCR have been describedelsewhere (8), and the cycling procedures were 41 cycles at 94 C, 50 C,and 72 C for 1 min each, followed by a 10-min extension at 72 C. ThePCR products were electrophoresed in 1.5% agarose, stained withethidium bromide, and visualized under ultraviolet transillumination.Chemotaxis detection by light microscopy. Freshly thawed

MGwas centrifuged to precipitate the organisms. After washing once, theMGwas resuspended in 400 ll of RPMI medium enriched with 10% calfserum. Boyden-type chemotactic chambers and previously describedprocedures were used to study the chemotactic activity of MG (5). To thebottom chambers was added 200 ll of PBS or 10�6 M of N-formyl-methionyl-leucyl-phenylalanine (fMLP) (Sigma Chemical Co., St.Louis, MO); 100 ll of MG was added to the top chambers. Betweenthe chambers was a 300-lm chemotactic membrane. fMLP is a knownchemoattractant that has the ability to attract the migration ofmacrophages, lymphocytes, and heterophils. After incubating the Boydenchamber for 60 min at 38 C, the membranes were removed and the topsurface of each membrane was wiped with an alcohol-soaked swab toremove organisms that were loosely attached. The membranes were thenfixed in 75% alcohol for 5 min and stained with Giemsa stain. Afterrinsing and drying, the membranes were mounted on a glass slide andexamined for the presence of bluish-stained cells at 4003magnification.Chemotaxis detection by fluorescence microscopy. The

thawed MG was washed once, resuspended in 100 ll of PBS and 20 llof 10 mg/ml of fluorescein isothiocyanate (FITC) (5), and incubated at38 C for 20 min. After washing once to remove unbound FITC, theMG was resuspended in RPMI medium enriched with 10% calf serum.The procedures for the chemotaxis assays were the same as those

152

AVIAN DISEASES 49:152–154, 2005

described in the previous section. To the bottom chambers was addedeither RPMI medium or fMLP, and to the top chambers was added100 ll of FITC-labeled MG. After incubation for 1 hr at 38 C, themembranes were removed, wiped, fixed in 75% alcohol for 5 min, airdried, mounted on a glass slide, and examined with an epifluorescencemicroscope for the presence of fluorescing cells.

Chemotaxis detection by scanning electron microscopy(SEM). The membranes were prepared as described for lightmicroscopy and were fixed in 2.5% glutaraldehyde and stored at 4 Cuntil SEM. The membrane has a clear side and a dull side. To examinefor the presence of cells on both sides, the membrane was sliced in halfin the middle; one half was mounted with the clear side up and the otherwith the dull side up. A Philips XL 30 scanning electron microscope(FEI Co., Hillsboro, OR) was used to visualize the presence of cells onthe membrane.

RESULTS AND DISCUSSION

Chemotaxis detection by fluorescence microscopy. Inthe control wells, a 0.8 6 0.6 (mean 6 SD) fluorescing MG cellswere detected in the chemotactic membranes, whereas in the fMLPwells 4.5 6 3.5 cells were detected. This indicates that in selected

fields a mean of 4.5 MG cells will respond and migrate into themembrane and to the location of the chemoattractant.

Chemotaxis detection by Giemsa stain. Fig. 1 shows thepresence of stained cells in a chemotactic membrane. In the controlwells, a mean 6 SD of 1.0 6 0.5 MG cells were detected in thechemotactic membranes, whereas in the fMLP wells 8.2 6 3.6 MGcells were detected. The sizes of the stained MG cells were largerthan the known size of MG cells, which is approximately 3 lm; thereason for this enlargement is unknown. In the two chemotaxisassays, the results indicate that MG can migrate into the membranesand can respond to chemoattractant fMLP and possibly to otherchemoattractants.

Chemotaxis detection by SEM. SEM was used to show thatMG can attach to and penetrate the chemotactic membranes only.Fig. 2A shows the presence of MG on the clear side of themembrane. Fig. 2B shows the presence of an organism approx-imately the size of MG on the dull side of the membrane, indicatingthat MG had penetrated to the other side of the membrane.

Chemotaxis detection by PCR. There was no amplifiableMG DNA in the allantoic fluids of the embryos harvested at 0, 1, 2,

Fig. 1. The presence of darkly stained MG in a chemotactic membrane. 4003.

Fig. 2. (A) The presence of MG on the clear side of the chemotactic membrane. (B) The presence of MG on the dull side of the chemotacticmembrane.

MG chemotaxis 153

3, 4, and 5 days after the deposition of MG on the shell membranes.The allantoic fluids harvested at 6 days or later had MG DNA thatwas amplifiable by the phenol-chloroform extraction method but notby the boiling method. The amplification was achieved only after 41cycles rather than the usual 31, indicating the presence of few copiesof MG DNA.While MG is known to attach to human cells (9), chicken red

blood cells (1,6,9), and chicken lymphocytes (6), there is noincontrovertible evidence that MG can be detected within the redblood cells or other cells. The results of the present study indicate thatMG can penetrate chemotactic membranes and eggshell membranes,providing preliminary evidence that MG can be detected within thecells. Because of the close proximity of the air sacs and the ovaries,these results raise the possibility that hens with MG-induced airsacculitis could transmit MG to the ovules by direct penetration ofthe membranes.

REFERENCES

1. Apostolov, K., and G. D. Windsor. The interaction of Mycoplasmagallisepticum with erythrocytes, I: morphology. Microbios 13:205–215.1975.

2. Baseman, J. B., M. Lange, N. L. Criscimagna, J. A. Giron, and C. A.Thomas. Interplay between mycoplasmas and host target cells. Microb.Pathog. 19:105–116. 1995.

3. Bredt, W. Motility. In : The mycoplasmas: cell biology, vol. 1. F.Barile and S. Razin, eds. Academic Press, New York. pp. 141–155. 1979.

4. Freundt, E. A. Culture media for classic mycoplasmas. In: Methods in

mycoplasmology, vol. 1. S. Razin and J. G. Tully, eds. Academic Press, New

York. pp. 127–135. 1983.5. Lam, K. M. A simple and fast way to count heterophils in chemotaxis.

Avian Dis. 42:812–814. 1998.6. Lam, K. M. Morphological changes in chicken cells after exposure to

Mycoplasma gallisepticum. Avian Dis. 48:488–493. 2004.7. Ley, D. H., and H. W. Yoder, Jr. Mycoplasma gallisepticum infection.

In: Diseases of poultry, 11th ed. Y. M. Saif, ed. Iowa State University Press,

Ames, IA. pp. 722–744. 2003.8. Nacimento, E. R., R. Yamamoto, K. R. Herrick, and R. C. Tait.

Polymerase chain reactions for detection of Mycoplasma gallisepticum. Avian

Dis. 35:62–69. 1993.9. Razin, S., M. Banal, H. Gamliel, A. Pollack, W. Bredt, and I.

Kahane. Scanning electron microscopy of mycoplasmas adhering to

erythrocytes. Infect. Immun. 30:538–546. 1980.10. Tajima, M., T. Nunoya, and T. Yagihashi. An ultrastructural study

on the interaction of Mycoplasma gallisepticum with the chicken tracheal

epithelium. Am. J. Vet. Res. 40:1009–1014. 1979.11. Taylor-Robinson, D., H. A. Davies, P. Sarathchandra, and P. M.

Furr. Intracellular location of Mycoplasma in culture cells demonstrated

by immunocytochemistry and electron microscopy. Int. J. Exp. Pathol. 72:

705–714. 1991.12. Uppal, P. K., and H. P. Chu. Attachment ofMycoplasma gallisepticum

to the tracheal epithelium of fowls. Res. Vet. Sci. 22:259–260. 1977.13. Winner, F., R. Rosengarten, and C. Citri. In vitro cell invasion of

Mycoplasma gallisepticum. Infect. Immun. 68:4238–4244. 2000.

154 K. M. Lam