HAL Id: hal-00490546
https://hal.archives-ouvertes.fr/hal-00490546
Submitted on 9 Jun 2010
HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.
L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.
invades non-phagocytic chicken cells
Daliborka Dušanić, Rebeka Lucijana Berčič, Ivanka Cizelj, Simona Salmič, Mojca Narat, Dušan Benčina
To cite this version:
Daliborka Dušanić, Rebeka Lucijana Berčič, Ivanka Cizelj, Simona Salmič, Mojca Narat, et al..
invades non-phagocytic chicken cells. Veterinary Microbiology, Elsevier, 2009, 138 (1-2), pp.114.
�10.1016/j.vetmic.2009.02.014�. �hal-00490546�
Accepted Manuscript
Title:Mycoplasma synoviaeinvades non-phagocytic chicken cellsin vitro
Authors: Daliborka Duˇsani´c, Rebeka Lucijana Berˇciˇc, Ivanka Cizelj, Simona Salmiˇc, Mojca Narat, Duˇsan Benˇcina
PII: S0378-1135(09)00104-7
DOI: doi:10.1016/j.vetmic.2009.02.014
Reference: VETMIC 4370
To appear in: VETMIC
Received date: 19-12-2008 Revised date: 17-2-2009 Accepted date: 20-2-2009
Please cite this article as: Duˇsani´c, D., Berˇciˇc, R.L., Cizelj, I., Salmiˇc, S., Narat, M., Benˇcina, D.,Mycoplasma synoviaeinvades non-phagocytic chicken cellsin vitro, Veterinary Microbiology(2008), doi:10.1016/j.vetmic.2009.02.014
This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Accepted Manuscript
Mycoplasma synoviaeinvades non-phagocytic chicken cellsin vitro 1
Daliborka Dušanića, Rebeka Lucijana Berčiča, Ivanka Cizelja, Simona Salmiča, Mojca 2
Narataand Dušan Benčinaa*, 3
a Department of Animal Science, Biotechnical Faculty, University of Ljubljana, 4
Groblje 3, 1230 Domžale, Slovenija 5
* Corresponding author. Tel.: +386 1 7217 809; fax: +386 1 7217 888. E-mail 6
address: [email protected](D. Benčina).
7
8
9
Benčina Dušan 10
Department of Animal Science 11
Biotechnical Faculty 12
University of Ljubljana 13
Groblje 3 14
1230 Domžale 15
Slovenija 16
Tel.: +386 1 7217 809 17
fax: +386 1 7217 888 18
E-mail address: [email protected] 19
* Manuscript
Accepted Manuscript
Abstract 21
Mycoplasma synoviae and Mycoplasma gallisepticum are major poultry pathogens, 22
but their strains differ significantly in invasiveness and pathogenicity. Recent studies 23
have demonstrated that M. gallisepticum invades chicken erythrocytes (CER) and 24
chicken embryonic fibroblasts. The aim of this study was to determine whether M.
25
synoviae also invades chicken cells. Using the gentamicin invasion assay, relative 26
invasion frequency (RIF) of four M. synoviae strains was determined for CER, 27
chicken embryonic cell line (CEC-32) and/or primary chicken chondrocytes (CCH).
28
All tested strains of M. synoviae were capable of invading chicken cells within 24 29
hours after infection. The type strain WVU 1853 showed significantly higher 30
invasiveness in CER (RIF 7.5 % ± 1.5) and CEC-32 (RIF 7.0 % ± 0.3) than field 31
strain ULB 02/T6 and M. gallisepticumstrain Rlow. Surprisingly, WVU 1853, which is 32
capable of causing synovitis and arthritis in chickens, was less invasive for CCH with 33
a RIF (1.2 % ± 0.3) similar to that of Rlow (1.1 % ± 0.1). This is the first study 34
documenting the invasiveness of M. synoviaestrains for non-phagocytic chicken cells.
35
Keywords: M. synoviae, M. gallisepticum, invasion, erythrocytes, fibroblasts, 36
chondrocytes 37
1. Introduction 38
Of the twelve recognized avian Mycoplasma species that infect chickens and/or 39
turkeys, M. gallisepticum and M. synoviae are the most pathogenic (Bradbury, 1998;
40
Kleven, 2003). The factors responsible for considerable differences in tissue tropism, 41
invasiveness and pathogenicity in strains of both species are largely unidentified.
42
Generally, in vitro passage of pathogenic microorganisms results in loss of 43
pathogenicity. This has been shown also for M. gallisepticum and M. synoviae 44
Accepted Manuscript
(Levisohn et al., 1986; Kleven, 2003). The low passage M. gallisepticum strain Rlow is 45
invasive and pathogenic for poultry, whereas invasiveness and pathogenicity 46
decreases in high passage strain Rhigh (Levisohn et al., 1986; Winner et al., 2000;
47
Much et al., 2003; Vogl et al., 2008). The factors contributing to the decreased 48
invasiveness of Rhigh strain are most likely the mutation(s) in the gene(s) encoding 49
cytadhesins and, possibly, decreased neuraminidase activity (NEAC) (Vogl et al., 50
2008; Berčič et al., 2008b). Sialic (neuraminic) acid is important in the process of 51
infection of host cells, because M. gallisepticum and M. synoviae adhere via 52
haemagglutinins to host-cell receptors containing sialic acid residues (Aldridge, 1975;
53
Razin, 1985; Benčina, 2002). In M. synoviae, a haemagglutination-positive (HA+) 54
phenotype has also been associated with a higher frequency of synovitis in chickens 55
inoculated into the hock joint (Narat et al., 1998).
56
The capacity to invade host cells has been demonstrated for fourMycoplasma species 57
known to be human pathogens (Lo et al., 1989; Baseman et al., 1995; Giron et al., 58
1996), as well as for poultry pathogen M. gallisepticum(Winner et al., 2000; Much et 59
al., 2003; Vogl et al., 2008). Studies have shown close association of M. synoviae 60
with chicken cells (Aldridge and Cole, 1978; Walker et al., 1978), but intracellular 61
presence of M. synoviaehas not been reported.
62
The main aim of this study was to determine whether M. synoviae is able to invade 63
non-phagocytic chicken cells. Using a well established method, the gentamicin 64
protection (invasion) assay, and three types of chicken cells, we assessed the invasion 65
frequency of different M. synoviaestrains and compared this with the invasiveness of 66
M. gallisepticumRlow. 67
2. Materials and methods 68
Accepted Manuscript
2.1 Chicken cells 69
Blood was taken from adult specific pathogen-free chickens and mixed with citrate 70
dextrose buffer (Sigma-Aldrich, Germany) in a 9:1 ratio. Erythrocytes (CER) were 71
harvested from the collected blood by adding sterile phosphate buffer saline (PBS) pH 72
7.4 and centrifugation at 400×g for 10 minutes. CER were washed in PBS, 73
centrifuged and resuspended in PBS to a final concentration of 5×105to 106cells/ml.
74
A stable transfected chicken embryonic fibroblast cell line (CEC-32) was donated by 75
Prof. Bernd Kaspers (University of Munich, Munich, Germany). CEC-32 cells 76
(Kaaden et al., 1982) were cultivated in Dulbecco's Modified Eagle Medium 77
(DMEM) supplemented with 8% fetal bovine serum (FBS) and 2% chicken serum (all 78
from Sigma-Aldrich, Germany).
79
For the isolation of chondrocytes from hyaline cartilage tissue, the protocol described 80
by Barličet. al. (2008) was modified. Briefly, cartilage tissue was obtained from adult 81
chicken tarsometatarsal joints and digested in DMEM containing 1mg collagenase 82
II/ml (Sigma-Aldrich, Germany) and 1µg gentamicin/ml (Krka, Slovenia) for 21 83
hours. The digested tissue was sieved through a 40 µm-pore nylon filter and the 84
chondrocytes (CCH) seeded into culture flasks containing DMEM with 8% FBS and 85
2% chicken serum. The viability of CCH after isolation, as shown by Trypan Blue 86
staining (Sigma-Aldrich, Germany), was 98 %. CCH were tested for mycoplasmas on 87
Frey’s agar and broth before the beginning of the study and in every experiment, and 88
were found to be mycoplasma-free. CCH and CEC-32 cells were cultured in a CO2
89
incubator at 38C in a 5% CO2atmosphere.
90
2.2 M. synoviae and M. gallisepticum cultures 91
Accepted Manuscript
M. synoviae reference strains WVU 1853 and ULB 02/T6, also used in previous 92
studies (Benčina et al., 2001; Lavrič et al., 2007; Berčič et al., 2008a), were used.
93
Recent field isolates M. synoviae ULB 08/T3 and ULB 08/T4 isolated from tracheas 94
of chickens were also used. The culture of invasive M. gallisepticumstrain Rlow, used 95
in previous studies by Winner et al. (2000) and Vogl et al. (2008) was donated by Dr.
96
Michael Szostak (University of Veterinary Medicine of Vienna, Vienna, Austria).
97
Cultures were grown in modified Frey’s medium with Bacto PPLO broth base (BD, 98
US) containing 12% porcine serum (Invitrogen, US), 800000 IU of benzylpenicillin 99
(Pliva, Croatia), glucose, vitamins and 0.1g of NAD/l (the latter three from Sigma- 100
Aldrich, Germany) at 38C (Kleven, 2003). The number of colony forming units 101
(CFU) was calculated as described previously (Rodwell and Whitcomb, 1983).
102
2.3 Haemadsorption, haemagglutination and adherence test 103
Haemadsorption (HAD) of CER to Mycoplasma colonies was determined as 104
described previously (Gardella and DelGuidice, 1983). A similar method was used to 105
test M. synoviae colonies for adsorption of CEC-32 cells and CCH. Briefly, agar 106
blocks (10 × 5 mm) bearing well-separated M. synoviaecolonies were overlaid with ~ 107
50 μl samples of PBS pH 7.4 containing 103-104CEC-32 cells or CCH. After 30 min 108
incubation at room temperature, blocks were gently washed with PBS. Binding of 109
CEC-32 cells and CCH was evaluated at 40× and/or 100× magnification.
110
Haemagglutination (HA) tests were performed as described previously (Narat et al., 111
1998; Benčina et al., 1999).
112
2.4 Sialic acid linkages of CEC-32 and CCH glycoproteins 113
To demonstrate the presence of sialic acid receptors potentially used by Mycoplasma 114
species to adhere to CEC-32 cells and CCH, proteins of both cell types were separated 115
Accepted Manuscript
by electrophoresis. The PhastGel (Gradient 8-25) and PhastSystem (Pharmacia, 116
Sweden), followed by transfer to Immobilon P membrane were used for CEC-32 117
cells, whereas the proteins of CCH were separated in a larger, 12 % polyacrylamide 118
gel (acrylamide mix, sodium dodecyl sulphate, ammonium persulphate and 119
tetramethylethylenediamine, all from Sigma-Aldrich, Germany) and then transferred 120
to Immobilon P membrane. The DIG Glycan Differentiation Kit (Roche, Slovenia) 121
was used according to the instructions of the manufacturer to identify CEC-32 cells 122
and/or CCH glycoprotein(s) containing sia(α-2-3) or sia(α-2-6) galactose linkages.
123
2.5 Determination of the minimal inhibitory concentration of gentamicin 124
The susceptibility of Mycoplasma strains for gentamicin was determined as the 125
minimal inhibitory concentration (MIC) of gentamicin in broth cultures of M.
126
synoviaestrains. MIC was determined as described previously (Hannan, 2000; Bebear 127
and Kempf, 2005). Briefly, 25 μl samples of rapidly growing M. synoviae cultures 128
(105-106 CFU/sample) were transferred into 1 ml of Frey´s broth without gentamicin 129
or into 1 ml of Frey’s broth supplemented with different concentrations of gentamicin.
130
For the gentamicin invasion assays, gentamicin was used in 500 μg/ml concentrations.
131
2.6 Experimental infection of chicken cells 132
CEC-32 cells and CCH were grown in 24-well culture plates until there were 133
approximately 5×105cells per well. The number of CER in samples used for infection 134
ranged from 5×105 to 106. CCH and CEC-32 cells had 98-100 % viability, as 135
determined by Trypan blue staining. Broth cultures of Mycoplasma strains used for 136
the infections were incubated till they reached the late logarithmic phase of growth.
137
100 µl of broth containing 107-108 CFU was then used to inoculate 1 ml (~5×105 138
cells) samples of chicken cells suspended in/overlayed with Frey’s broth. Thus, the 139
Accepted Manuscript
multiplicity of infection (MOI) ranged from 10 to 100 mycoplasmas per chicken cell.
140
The infection of cells was performed at 37-38C. Uninfected samples of appropriate 141
chicken cells were used as negative controls. In all experiments, the frequency of 142
invasion (RIF) was assayed 24 hours after infection. In some experiments, the RIF 143
after 2-4 hours and/or after 48 hours was also determined.
144
2.7 Gentamicin invasion assay 145
The number of intracellular mycoplasmas in CER, CEC and CCH was determined 146
using a modified gentamicin invasion assay described by Winner et al. (2000) and 147
Vogl et al. (2008). Briefly, 2-4, 24 or 48 hours after infection, the whole contents of 148
each well were centrifuged for 10 min at 300×g. The pellets were then resuspended 149
either in 1 ml of Frey’s broth without gentamicin, or in 1 ml of Frey’s broth 150
supplemented with 500µg of gentamicin/ml. Cells were resuspended 8 times during 151
the 4 hours of incubation at 38C in 5% CO2. After treatment, the cell suspensions 152
were washed three times with PBS. The infected pelleted cells treated with 153
gentamicin were resuspended in approximately 150 μl of Frey’s broth and 50 μl 154
samples were plated onto Frey´s agar and incubated at 38C. The infected cells, which 155
were not treated with gentamicin were resuspended in 1.5 ml of Frey’s broth and 156
plated as described above. Uninfected cell monolayers incubated in Frey’s broth in 157
the same way as the infected cells were used as negative controls. After 5-7 days, 158
mycoplasma colonies on the plates were identified and counted as described in section 159
2.8. The relative invasion frequency (RIF) was calculated as the percentage of the 160
number of mycoplasma colonies recovered after gentamicin treatment relative to the 161
number of colonies recovered from samples that were not exposed to gentamicin 162
(Vogl et al. 2008). Data from independent assays, that were performed in triplicates, 163
Accepted Manuscript
were expressed as means ± S.E. The significance of the differences between RIF of 164
the strains tested in one cell line and between cell lines infected with the same strain 165
of mycoplasmas were assessed using Student’s unpaired t-test. Differences were 166
considered significant for pvalues under 0.05.
167
2.8 Identification of colonies 168
M. gallisepticum and M. synoviae colonies were identified using the indirect 169
immunoperoxidase assay (IIPA) and/or direct immunofluorescence as described 170
previously (Benčina et al., 1994; Benčina and Bradbury, 1992). The FITC-conjugated 171
antibodies to M. synoviae used for the direct immunofluorescence (Supplementary 172
Fig. 1), were also used in the differential immunofluorescent-antibody test to identify 173
intracellular M. synoviae(Supplementary Figs. 2 and 3).
174
2.9 Differential immunofluorescence microscopy 175
Double immunofluorescent-antibody test (Heesemann and Laufs, 1985) was modified 176
as follows. Differentially labeled rabbit antibodies specific to M. synoviae (Benčina 177
and Bradbury, 1992) were used for detecting M. synoviaeon the surface and/or inside 178
chicken cells in a direct immunofluorescent assay. Cells were fixed in 4 % PBS- 179
buffered paraformaldehyde for 40 min. Antibodies conjugated with tetramethyl 180
rhodamine isothiocyanate (TRITC) (diluted 1:100, 1 h at room temperature) were 181
used for detecting M. synoviae on the surface of chicken cells. Following washing in 182
PBS, cells were permeabilised with 1 % Triton X-100 (Sigma-Aldrich, Germany) in 183
PBS for 10 min and incubated in 1 % bovine serum albumin in PBS for 30 min.
184
Antibodies conjugated with fluorescein isothiocyanate (FITC) (diluted 1:100, 1 h at 185
room temperature) were used for immunostaining of intracellular M. synoviae. Dako 186
Fluorecence Mounting medium (Dako, USA) was used to prevent fluorescence 187
Accepted Manuscript
fading. Fluorescence microscopy was performed using a Carl Zeiss LSM 510 188
confocal microscope. Images were analyzed using Carl Zeiss LSM image software 189
190 3.0.
3. Results 191
3.1 Adherence of M. synoviae to chicken cells used in the invasion assays 192
Colonies of all the M. synoviae and M. gallisepticum Rlow cultures used were 193
predominately HAD+, but all cultures also contained some colonies that did not 194
adsorb chicken erythrocytes (HAD-). The pattern of adsorption of CCH and CEC-32 195
cells to colonies of M. synoviae was similar to that observed with erythrocytes. Strain 196
ULB 02/T6 had a higher HA titre than other M. synoviae strains (Table 1).
197
Examination of CEC-32 cells and CCH using the DIG Glycan Differentiation Kit 198
showed presence of glycoproteins containing sia(α2-3)gal and sia(α2-6)gal linkages.
199
CCH showed a major sialylated glycoprotein of about 70 kDa (Fig. 1), while CEC-32 200
cells contained a major sialylated glycoprotein of about 60 kDa (data not shown).
201
3.2 Invasion of chicken erythrocytes 202
The RIF for chicken erythrocytes was determined for two M. synoviaestrains (WVU 203
1853 and ULB 02/T6) and compared to the RIF for M. gallisepticum strain Rlow
204
(Tables 1,2), which invades sheep and chicken erythrocytes (Vogl et al., 2008). M.
205
synoviae strains were approximately 20-fold more susceptible to gentamicin than M.
206
gallisepticumRlow(Table 1).
207
Three hours after infection, M. gallisepticumand both M. synoviaestrains were viable 208
in erythrocytes treated with gentamicin, most probably because they had established 209
intracellular residence. However, their estimated RIF values after 3 hours were much 210
Accepted Manuscript
lower (5- to 10-fold) than after 24 hours. After 24 hours, the mean RIF for M.
211
gallisepticum Rlow (1.2 % ± 0.7) was similar to that reported by Vogl et al. (2008).
212
The mean RIF for theM. synoviae strain ULB 02/T6 was also similar (Table 2). The 213
type strain ofM. synoviae, WVU 1853, had the greatest invasiveness for CER with a 214
mean RIF (7.5 % ± 1.5) more than 6-fold higher than that of M. gallisepticum Rlow
215
(Table 2). All three mycoplasma strains were capable of surviving within CER for 48 216
hours, but with no increase in the RIF after 24 hours for any strain. Invasion of CER 217
was confirmed by immunofluorescent staining of internalized M. synoviae, which 218
reacted with specific antibodies labeled with FITC after permeabilization of CER 219
(data not shown).
220
3.3 Invasion of CEC-32cells 221
Invasion of CEC-32 cells by M. gallisepticum Rlow was detected 4 hours after 222
infection. After 24 hours, the mean RIF was 1.0 % ± 0.2 and did not increase over the 223
following 24 hours (Table 2, data for 4 and 48-hour infection not shown).M. synoviae 224
strains WVU 1853 and ULB 02/T6 also invaded CEC-32 cells after 4 hours of 225
infection and survived inside them for 48 hours. However, after 24 hours of infection, 226
the mean RIF of WVU 1853 (7.0 % ± 0.3) was almost 6-fold higher in comparison to 227
ULB 02/T6. Lethality for CEC-32 cells was not observed over the first 24 hours (data 228
not shown). Differential immunofluorescence microscopy confirmed the invasion of 229
CEC-32 cells with WVU 1853 24 hours after infection. Besides numerous 230
extracellular M.synoviae located at CEC-32 cells’ surface, there were also distinct 231
green foci inside these cells indicating the intracellularM. synoviaecells, which were 232
immunostained only with antibodies conjugated with FITC. Superimposed images of 233
micrographs showing red and green fluorescence showed extracellular M. synoviae as 234
yellow foci or aggregates, whereas the presence of intracellular M. synoviae was 235
Accepted Manuscript
indicated by green fluorescence (Supplementary Fig. 2). Confocal scan of CEC-32 236
cells infected with WVU 1853, shown in Supplementary Fig. 3, confirmed the 237
intracellular localization of M. synoviae. M. synoviae labeled with FITC-conjugated 238
antibodies were located in the cytoplasm of CEC-32 cells and became visible when 239
the image was scanned from the top to the bottom.
240
3.4 Invasion of chicken chondrocytes 241
After 24 hours of infection, RIF for the M. gallisepticumstrain Rlowin CCH was quite 242
similar (1.1 % ± 0.1) to that seen in CER and CEC-32 cells, as well as to that ofM.
243
synoviae strain WVU 1853 (1.2 % ± 0.3) in CCH (Table 2). On the other hand, the 244
invasiveness of WVU 1853 was 6-fold lower in CCH than in CER and CEC-32 cells.
245
The capability to invade CCH within 2 hours after infection was determined for WVU 246
1853 (RIF ~ 0.25 %) and field strains ULB 08/T3 and ULB 08/T4. The latter two 247
strains were used at a low passage, but after 24 hours of infection their RIF was not 248
higher than that of WVU1853 (Table 2). M. synoviae strains remained viable within 249
CCH for 48 hours after infection, but there was no evidence of further increase of 250
their RIF (data not shown). The ability of WVU 1853 to invade CCH in 24 hours after 251
infection was confirmed by differential immunofluorescence microscopy. In 252
agreement with the gentamicin invasion assay results, the extracellular population of 253
M. synoviaewas predominant and appeared to form small aggregates on the surface of 254
CCH. However, distinct foci of green fluorescence inside CCH, which were present 255
only in CCH infected with M. synoviae, confirmed that it invades CCH (data not 256
shown).
257
4. Discussion 258
Accepted Manuscript
Although M. gallisepticum and M. synoviae belong to different phylogenetic groups, 259
they share hosts, tissue tropism and a number of horizontally transferred genes 260
(Noormohammadi et al., 1998; Berčič et al., 2008a). Recent studies have provided 261
evidence that some M. gallisepticum strains can invade cells, including chicken 262
embryonic fibroblasts and erythrocytes (Winner et al., 2000; Vogl et al., 2008). This 263
is the first study to show that M. synoviae can invade chicken erythrocytes, 264
chondrocytes and embryonic cells in vitro.
265
The well established gentamicin invasion assay, previously used to demonstrate 266
invasion by M. gallisepticum (Winner et al., 2000; Vogl et al., 2008) was used with 267
infected chicken cells exposed to 500 μg gentamicin/ml, a concentration almost 200- 268
fold higher than the MIC for the M. synoviae strains tested. The M. synoviae strains 269
used were at least 20-fold more susceptible to gentamicin than M. gallisepticum Rlow
270
(Table 1). This is consistent with the data published for MIC of these Mycoplasma 271
species (Bebear and Kempf, 2005). Thus, it is very likely that all M. synoviae 272
recovered from the infected chicken cells originated from M. synoviae cells that had 273
invaded the chicken cells and thus escaped the mycoplasmacidal effect of gentamicin.
274
All M. synoviaecultures recovered from CER, CCH and CEC-32 cells after treatment 275
with gentamicin had the same MIC as the original cultures, between 2 and 3 μg/ml.
276
It has been suggested that the HAD+phenotype of M. gallisepticum strain Rlowcould 277
be responsible for its higher invasiveness in erythrocytes (Vogl et al., 2008). In this 278
study, the type strain WVU 1853 of M. synoviaehad the highest RIF in erythrocytes, 279
but another M. synoviae strain (ULB 02/T6) had a higher HA titre (Tables 1 and 2).
280
Thus, there must be additional factors that contribute to invasiveness in erythrocytes, 281
as well as CEC-32 cells and CCH. These factors may differ for different host cells as 282
WVU 1853 had about 6-fold higher RIF in CER and CEC-32 cells than in CCH.
283
Accepted Manuscript
The adherence to chicken cells via receptors containing sialic acid is important in 284
infections with M. gallisepticum and M. synoviae (Aldridge, 1975; Razin, 1985;
285
Benčina, 2002). It is likely that major sialylated glycoprotein(s) of CCH (~ 70 kDa) 286
(Figure 1) and CEC-32 cells (~ 60 kDa) could be involved in binding of M. synoviae 287
and M. gallisepticum to these chicken cells. However, further investigations are 288
required to confirm this and to identify genes encoding sialylated glycoproteins of 289
CEC-32 and CCH cells.
290
It is probable that the gentamicin invasion assay indicates the number of infected host 291
cells better than the number of invasive mycoplasmas (Vogl et al., 2008). Differential 292
immunofluorescent staining showed that CER and CEC-32 cells were mostly infected 293
with M. synoviaeattached to the surface these cells, but that a considerable number of 294
cells contained intracellular M. synoviae(Supplementary Figs. 2 and 3). Although the 295
invasion frequency was not estimated with immunofluorescence microscopy, this 296
finding confirms the capacity of M. synoviaeto invade chicken cells in vitro.
297
It remains to be proved that M. synoviae also invades chicken cells in vivo. Infections 298
of the upper respiratory tract with M. synoviae may be lifelong and are usually 299
detectable by culturing swabs of the upper part of trachea and/or choanal cleft 300
(Kleven, 2003). Intracellular localization could help explain, at least partially, 301
persistence of M. synoviae despite of specific local antibodies and phagocytosis and 302
antibiotic treatment.
303
5. Conclusion 304
This study provides the first evidence for invasion and intracellular survival of M.
305
synoviaein three different non-phagocytic chicken cell types in vitro. These findings 306
Accepted Manuscript
contribute to understanding of how M. synoviae evades immune surveillance and 307
persists in immunocompetent chickens.
308
6. Acknowledgements 309
This work was supported by grant 1000-07-310121 from the Ministry of Education 310
and Science of the Republic of Slovenia. For erythrocytes from SPF-chickens, we 311
thank Prof. Dr. Olga Zorman Rojs. For the donation of Mycoplasma gallisepticum 312
strain Rlow, we thank Dr. Michael Szostak and for the donation of CEC-32 cells Prof.
313
Bernd Kaspers. We would also like to thank Drs. Irena Oven and Nataša Obermajer 314
for the help with the preparation of fluorescence images.
315
7. References 316
Aldridge, K.E., 1975. Growth and cytopathology of Mycoplasma synoviae in chicken 317
embryo cell culture. Infect. Immun. 12, 198-204.
318
Aldridge, K.E., Cole, B.C., 1978. Immunofluorescence and electron microscopy of 319
the attachment of Mycoplasma synoviae to chicken embryo fibroblasts. Infect.
320
Immun. 21, 328-332.
321
Barlič, A., Drobnic, M., Malicev, E., Kregar-Velikonja N., 2008. Quantitative 322
analysis of gene expression in human articular chondrocytes assigned for 323
autologous implantation. J. Orthop. Res. 26, 6, 847-853.
324
Baseman, J.B., Lange, M., Criscimagna, N.L., Giron, J.A., Thomas, C.A., 1995.
325
Interplay between mycoplasmas and host target cells. Microb. Pathog. 19, 2, 105- 326
327 116.
Accepted Manuscript
Bebear, C.M., Kempf, I., 2005. Antimicrobial therapy and antimicrobial resistance.
328
In: Blanchard, A., Browning, G. (Eds.), Mycoplasmas Molecular biology, 329
pathogenicity and strategies for control, Horizon bioscience, pp. 535-569.
330
Benčina D., 2002. Haemagglutinins of pathogenic avian mycoplasmas. Avian Pathol.
331
31, 6, 535-547.
332
Benčina, D., Bradbury, J.M., 1992. Combination of immunofluorescence and 333
immunoperoxidase techniques for serotyping mixtures of Mycoplasma species. J.
334
Clin. Microbiol. 30, 407-410.
335
Benčina, D., Kleven, S.H., Elfaki, M.G., Snoj, A., Dovč, P., Dorrer, D., Russ, I., 336
1994. Variable expression of epitopes on the surface of Mycoplasma gallisepticum 337
demonstrated with monoclonal antibodies. Avian Pathol. 23, 19-36.
338
Benčina, D., Narat, M., Dovc, P., Drobnič-Valič, M., Habe, F., Kleven, S. H., 1999.
339
The characterization of Mycoplasma synoviae EF-Tu protein and proteins 340
involved in hemadherence and their N-terminal amino acid sequences. FEMS 341
Microbiol. Lett. 173, 85-94.
342
Benčina, D., Drobnič-Valič, M., Horvat, S., Narat, M., Kleven, S.H., Dovč, P., 2001.
343
Molecular basis of the length variation in the N-terminal part of Mycoplasma 344
synoviaehemagglutinin. FEMS Microbiol. Lett. 203, 115-123.
345
Berčič, R.L., Slavec, B., Lavrič, M., Narat, M., Bidovec, A., Dovč, P., Benčina, D., 346
2008a. Identification of major immunogenic proteins of Mycoplasma synoviae 347
isolates. Vet. Microbiol. 127, 147-154.
348
Accepted Manuscript
Berčič, R.L., Slavec, B., Lavrič, M., Narat, M., Zorman-Rojs, O., Dovč, P., Benčina, 349
D., 2008b. A survey of avian Mycoplasma species for neuraminidase enzymatic 350
activity. Vet. Microbiol. 130, 391-197.
351
Bradbury, J.M., 1998. Recovery of mycoplasmas from birds. In: Miles, R., Nicholas, 352
R. (Eds.), Mycoplasma protocols, Humana Press Totowa, New Jersey, pp. 45-54.
353
Gardella, R.S., DelGuidice, R.A., 1983. Hemagglutination, Hemadsorption, and 354
Hemolysis. In: Razin, S., Tully, J.G. (Eds.), Methods in mycoplasmology I 355
Mycoplasma characterization, Academic Press, pp. 379-396.
356
Giron, J.A., Lange, M., Baseman, J.B., 1996. Adherence, fibronectin binding and 357
introduction of cytoskeleton reorganization in cultured human cells by 358
Mycoplasma penetrans. Infect. Immun. 64, 197-208.
359
Hannan, P.C.T., 2000. Guidelines and recommendations for antimicrobial minimum 360
inhibitory concentration (MIC) testing against veterinary Mycoplasma species.
361
Vet. Res. 32, 373-395.
362
Heeseman, J., Laufs, R., 1985. Double immunofluorescence microscopic technique 363
for accurate differentiation of extracellularly and intracellularly located bacteria in 364
cell culture. J. Clin. Microbiol. 22, 2, 168-175.
365
Kaaden, O.R., Lange, S., Stiburek, B., 1982. Establishment and characterization of 366
chicken embryo fibroblast clone LSCC-H32. In Vitro., 18, 10, 827-834.
367
Kleven, S.H., 2003. Mycoplasma synoviae infection. In: Saif, Y.M., Barnes, H.J., 368
Glisson, J.R., Fadly, A.M., McDougald, L.R., Swayne, D.E. (Eds.), Diseases of 369
poultry 11thed., Iowa State University Press, Ames, pp. 756-766.
370
Accepted Manuscript
Lavrič, M., Benčina, D., Kothlow, S., Kaspers, B., Narat, M., 2007. Mycoplasma 371
synoviaelipoprotein MSPB, the N-terminal part of VlhA haemagglutinin, induces 372
secretion of nitric oxide, IL-6 and IL-1beta in chicken macrophages. Vet.
373
Microbiol. 15, 121, 278-287.
374
Levisohn, S., Dykstra, M.J., Lin, M.Y., Kleven, S.H., 1986. Comparison of in vivo 375
and in vitro methods for pathogenicity evaluation for Mycoplasma gallisepticum 376
in respiratory infection. Avian Pathol. 15, 233-246.
377
Lo, S.C., Dawson, M.S., Wong, D.M., Newton III, P.B., Sonoda, M.A., Engler, W.F., 378
Wang, R.Y., Shih, J.W., Alter, H.J., Wear, D.J., 1989. Identification of 379
Mycoplasma incognitus infection in patients with AIDS: an 380
immunohistochemical, in situhybridization and ultrastructural study. Am. J. Trop.
381
Med. Hyg. 41, 601-616.
382
Much, P., Winner, F., Stipkovits, L., Rosengarten, R., Citti, C., 2003. Mycoplasma 383
gallisepticum: influence of cell invasiveness on the outcome of experimental 384
infection in chickens. FEMS Immunol. Med. Microbiol. 34, 181-186.
385
Narat M., Benčina, D., Kleven, S.H., Habe, F., 1998. The hemagglutination-positive 386
phenotype of Mycoplasma synoviae induces experimental infectious synovitis in 387
chickens more frequently than does the hemagglutination-negative phenotype.
388
Infect. Immun. 66, 12, 6004-6009.
389
Noormohammadi, A.H., Markham, P.F., Duffy, M.F., Whithear, K.G., Browning, 390
G.F., 1998. Multigene families encoding the major hemagglutinins in 391
phylogenetically distinct mycoplasmas. Infect Immun. 66, 7, 3470-3475.
392
Accepted Manuscript
Razin, S., 1985. Mycoplasma adherence. In: Razin, S., Barile, M.F. (Eds.), The 393
Mycoplasmas IV Mycoplasma Pathogenicity, Academic Press, pp. 160-202.
394
Rodwell, A.W., Whitcomb, R.F., 1983. Methods for direct and indirect measurement 395
of mycoplasma growth. In: Razin, S., Tully, J.G. (Eds.), Methods in 396
mycoplasmology I, Academic Press, pp. 185-196.
397
Vogl, G., Plaickner, A., Szathmary, S., Stipkovits, L., Rosengarten, R., Szostak, M.P., 398
2008. Mycoplasma gallisepticum invades chicken erythrocytes during infection.
399
Infect. Immun. 76, 1, 71-77.
400
Walker, E.R., Friedman, M.H., Olson, N.O., Sahu, S.P., Mengoli, H.F., 1978. An 401
ultrastructural study of avian synovium infected with an arthrotropic Mycoplasma, 402
Mycoplasma synoviae. Vet. Pathol. 15, 3, 407-416.
403
Winner, F., Rosengarten, R., Citti, C., 2000. In vitro cell invasion of Mycoplasma 404
gallisepticum. Infect. immun 68, 7, 4238-4244.
405
406
407
Accepted Manuscript
Table 1: Mycoplasmaspecies, their strains and chicken cells used in the gentamicin invasion assay a 408
Chicken cells e Species Strain Passages b
MIC (gentamicin)
(μg/ml)c HA titre (×103)d CER CEC-32 CCH References
M. gallisepticum Rlow 20 50-80 NAf 3 3 2 Vogl et al., 2008
M. synoviae WVU 1853 >20 2-3 1.6 3 3 4 Benčina et al., 2001
M. synoviae ULB 02/T6 10 2-3 12.8 3 3 NA Berčič et al., 2008a
M. synoviae ULB 08/T3 2 2-3 6.4 NA NA 4g This study
M. synoviae ULB 08/T4 2 2-3 3.2 NA NA 3 This study
409
aSamples of chicken cells infected with mycoplasmas were divided into two parts. One was exposed to gentamicin (500 μg/ml) for4 h, whereas the other was 410
not (see Materials and Methods).
411
bNumbers of in vitro passages before an appropriate mycoplasma culture was used for gentamicin invasion assay in this study.
412
cMinimal inhibitory concentration of gentamicin (μg/ml).
413
dHaemagglutination titer, the reciprocal of the highest dilution of mycoplasmal cell pellet which gave complete HA of 1 % suspension of chicken erythrocytes 414
(Narat et al., 1998).
415
e CER: chicken erythrocytes, CEC-32: chicken embryonic cells, CCH: chicken chondrocytes. The numbers indicate the number of independent assays 416
performed.
417
f NA: not assayed.
418
gResults of only three experiments were shown in Table 2.
419
Accepted Manuscript
Table 2: Relative invasion frequency of M. gallisepticumand M. synoviaestrains for 420
chicken cellsa 421
Species
% of invasion for chicken cells b
Strain CER CEC-32 CCH
M. gallisepticum Rlow 1.2 ± 0.7 1.0 ± 0.2 1.1 ± 0.1 M. synoviae WVU 1853c 7.5 ± 1.5 7.0 ± 0.3 1.2 ± 0.3
M. synoviae ULB 02/T6 1.4 ± 1.1 1.2 ± 0.5 NA
M. synoviae ULB 08/T3 NAd NA 0.6 ± 0.2e
M. synoviae ULB 08/T4 NA NA 0.7 ± 0.2
422
aEvaluated and calculated 24 h after infection of appropriate cells with mycoplasmas (MOI 423
ranged from 10 to 100 mycoplasmas per chicken cell).
424
bEvaluated and calculated as described in Materials and Methods (see also Vogl et al. 2008).
425
cThe difference in invasiveness of M. synoviae strain WVU 1853 was significant for CER 426
versus CCH (p < 0.003) and for CEC-32 versus CCH (p ≤ 0.0001).The difference between 427
RIF of M. synoviae strain WVU 1853 and both the M. synoviae ULB02/T6 and M.
428
gallisepticumRlowwas also significant for both CER (p < 0.005 and <0.003, respectively) and 429
CEC-32 (p ≤ 0.0001 for both Mycoplasmaspecies). There was no significance between the 430
differences in RIF of Mycoplasmaspecies for CCH.
431
dNA: not assayed 432
eExtremely high RIF in one experiment (9.25 %) is not included in the mean RIF for ULB 433
08/T3. It was about one order of magnitude higher than all other RIF values in CCH in this 434
study and was considered erroneous.
435 436
437
438
439
440
441
442
Accepted Manuscript
Figure captions 443
Figure 1: Major sialylated glycoprotein of chicken chondrocytes. Proteins were 444
separated by SDS-PAGE, transferred to ImmobilonP membrane and analyzed for 445
sialylation using DIG Glycan Differentiation Kit. MAA: proteins binding the Maackia 446
amurensis agglutinin, which detects sia(α2-3)gal linkages. GAPDH (glyceraldehyde 447
3P dehydrogenase), indicated by arrow, was labeled using specific monoclonal 448
antibodies by additional immunostaining. SNA: proteins binding to Sambucus nigra 449
agglutinin, which recognises sia(α2-6) gal linkages. MWM: Molecular weight marker.
450
Supplementary Figure captions 451
Figure 1: Identification of the Mycoplasma synoviae WVU 1853 colonies by direct 452
immunofluorescence. Panel A shows native WVU 1853 colonies isolated from CCH 453
(24-hour infection) treated with gentamicin. Panel B shows the same colonies of 454
WVU 1853 that were immunostained with specific antibodies labeled with FITC. The 455
same FITC-conjugated antibodies were used to detect intracellular M. synoviae in 456
infected chicken cells (Supplementary Figs. 2 and 3).
457
Figure 2: Differential immunofluorescence images of a CEC-32 cell infected with M.
458
synoviaeWVU 1853 for 24 hours. The CEC-32 cells infected with WVU 1853 were 459
incubated with specific antibodies labeled with TRITC and, following 460
permeabilisation, with specific antibodies labeled with FITC. The same area of 461
confocal microscopic image was analyzed for extracellular M. synoviae labeled with 462
TRITC (red fluorescence, panel A) and for extra- and intracellular M. synoviae(green 463
fluorescence, panel B). Superimposition of the red and green fluorescence (panel C) 464
resulted in yellow fluorescence, indicating extracellular M. synoviae, whereas the 465
green fluorescent foci indicated M. synoviaeinside infected CEC-32 cells.
466
Accepted Manuscript
Figure 3: Confocal scan of a CEC-32 cell infected with M. synoviaeWVU 1853 after 467
differential immunofluorescence staining. The same area of confocal microscopic 468
image was scanned to identify intracellular M. synoviaelabeled with FITC-conjugated 469
antibodies (green fluorescence). In each panel, images are superimpositions of the red 470
and green fluorescence. A: Scattered points of green fluorescence indicate presence of 471
M. synoviae. Following moving of the scan downward (for 0.82 μm), intracellular 472
M.synoviae cells labeled with FITC-conjugated antibodies begin to appear as 473
aggregates emitting bright green fluorescence (B). C: Moving of the scan further 474
downward reveals M. synoviaecells, that reside exclusively inside the CEC-32 cell.
475
476
Accepted Manuscript
MWM MAA SNA
95
72
55
43
34
Fig. 1: Major sialylated glycoprotein of chicken chondrocytes. Proteins were separated by
SDS-PAGE, transferred to ImmobilonP membrane and analyzed for sialylation using DIG Glycan Differentiation Kit. MAA: proteins binding the Maackia amurensisagglutinin, which detects sia(α2-3)gal linkages. GAPDH (glyceraldehyde 3P dehydrogenase), indicated by arrow, was labelled using specific monoclonal antibodies by additional immunostaining.
SNA: proteins binding to Sambucus nigraagglutinin, which recognises sia(α2-6) gal linkages.
MWM: Molecular weight marker.
GAPDH Figure 1
