BACKGROUND
in  <dig>  mycoplasma and cell cultures were first associated in laboratory contamination  <cit> .
this contamination affects research by invalidating results in diagnosis.
however interference by these bacteria in mammalian non phagocytic cell cultures has been used to study mollicute biology  <cit> .
the opportunism of mollicutes is a challenging subject.
these microbes are diverse enough to explain their relationship variety with the host cells  <cit> .
the adhesion seems crucial for their pathogenicity  <cit> .
in addition, some mollicutes have been detected inside non naturally phagocytic cells.
in fact, the intracellular location is well protected from the immune system and some antibiotics  <cit> .
the use of non-phagocytic cells to study mollicutes has been of great interest mainly since mycoplasma fermentans was initially considered a cofactor in the pathogenesis of aids  <cit> .
other mycoplasmas showed this same characteristic when inoculated in non-phagocytic cells such as m. fermentans  <cit> , m. pneumoniae  <cit> , m. genitalium  <cit>  and m. gallisepticum  <cit> .
ureaplasma diversum is a bovine-originated mollicute, first isolated in  <dig> and considered a non-pathogenic species.
although detected in healthy animals, it is currently considered a pathogenic species due to its strong association with cattle diseases such as placentitis, fetal alveolitis, abortion and birth of weak calves  <cit> .
as with most animal mycoplasmosis, the cause of ureaplasma-associated reproductive disease is multifactorial  <cit> .
in bulls, this ureaplasma is an important pathogen of the genital tract, involved in such diseases as lowered sperm motility, seminal vesiculitis, and epididymitis  <cit> .
nevertheless, little is known about the virulence and pathogenic mechanisms of this mollicute.
because the invasion of u. diversum in not known, we inoculated this mollicute in hep- <dig> cells and observed this infection through confocal laser scanning microscopy  and used a gentamicin invasion assay.
RESULTS
u. diversum adhesion and invasion on hep- <dig> cells observed by clsm
the images of infected cells were from the apical surface to the basolateral region and differentiated the actin filaments in green, from the blue luminescence of nuclei.
therefore the ureaplasmas were detected in red luminescence, discriminating their arrangements in the serial sections of the infected cells.
the dil solution did not show ureaplasmal cytotoxicity  and allowed for differentiating the hep- <dig> cells from ureaplasmal arrangements.
non-infected hep- <dig> cells did not exhibit distinct intracellular dil fluorescence.
the images obtained showed adhesion and invasion of u. diversum in hep- <dig> cells .
after one minute of infection, a few ureaplasmal cells were detected scattered and inside the hep- <dig> cells .
after  <dig> minutes of infection, the ureaplasma persists, scattered, but increased inside the hep- <dig> cells .
at  <dig>   <dig> and  <dig> hours of infection, the microorganisms were detected mostly surrounding the perinuclear regions .
the studied microorganisms showed no differences in their distribution when adhered to or inside the cytoplasm after  <dig> hours of infection.
ureaplasmal infection produced no cytopathic effects in hep- <dig> cells in the studied period.
disposal of u. diversum in the infected hep- <dig> cells
gentamicin invasion assay
the ub medium promoted the growth of studied ureaplasmas.
the exposure of inoculum size of ureaplasmas used for gentamicin allowed no recovery in ub medium.
however the ureaplasma of infected hep- <dig> cells incubated with gentamicin and trypsinized allowed recovery of this microorganism.
in this assay, it was possible to determine that the clinical isolates of ureaplasma revealed to be more concentrated in hep- <dig> cells than reference strains.
this quantification was determined by 10-fold dilutions of ureaplasma obtained after gentamicin assay in ub medium and expressed as changing color units/ml .
therefore, the internalization of studied ureaplasma in hep- <dig> was confirmed and quantified in this assay.
gentamycin is impermeable to mammalian cells in the concentration used: it kills only the extra cellular ureaplasma but not the internalized bacteria.
the rates of invasion were expressed as the percentage of ccu obtained after antibiotic exposure relative to the initial inoculum .
the calculated p-value <  <dig> e- <dig>  test for equality of proportions with continuity correction, r project, vienna, austria allow for concluding that approximately 1% of the initial inoculum had survived the gentamicin treatment in type-strains and about 10% in clinical isolates.
the atcc strain has a high passage in ub medium.
no differences were observed in frequency of invasion between high and low passages clinical isolates .
phospholipase c activity
the ureaplasmas were initially cultured at 37°c for  <dig> hours in one ml of ub broth with pnppc.
the supernatants were evaluated at a wavelength of  <dig> nm  in a multiskan microplate reader .
the phospholipase c activity was found in the studied ureaplasma and all produced high levels of this enzyme.
the average activity was  <dig>  to  <dig>  pnppc hydrolysis  .
this was the highest level that allowed detection of this compound in the present study.
the phospholipase c activity also measured in sonicated ureaplasmas cells.
the average activity was  <dig>  to  <dig>  pnppc hydrolysis .
these results showed that most activity is related to secreted enzyme.
no differences were detected between the reference strains and clinical isolates.
however this activity could be associated with a feature for invasion of ureaplasma.
discussion
adhesion and invasion has been studied in a few mollicutes, most being human-originated species.
adhesion is considered an important feature to pathogenesis of these bacteria, and the invasion, a subsequent event, has been described in phagocytic or non phagocytic cells.
therefore chronic and recurrent mycoplasmosis may be explained in part by the reported failures of antibiotic treatments and immune response escape  <cit> .
vancini & benchimol  <cit>  reported m. hominis invasion in trichomonas vaginalis and escaped from the vacuolization of trichomonad cytosol.
this finding adds to understanding the challenging features of mollicute biology and their transmission among the hosts.
consistent with other studied mollicutes, the infection described herein with u. diversum in hep- <dig> cells allowed for identifying this ureaplasma as another mammalian cell invader and may also explain and support prior findings on some ureaplasmal infections in bovines.
cslm has been used to detect mollicute invasion in non phogocytic cells confirming its advantage in detecting u. diversum invasion.
the gentamicin invasion assay also confirmed this finding.
u. diversum was detected in hep- <dig> cells one minute after infection.
m. penetrans has been observed as early as  <dig> minutes after infection in hela cells  <cit> , while in hep- <dig> cells, the invasion occurred after  <dig> hours of infection  <cit> .
cell internalization after  <dig> minutes was also detected for m. genitalium in hela cells, and the mycoplasmas remained inside the cells for  <dig> days  <cit> .
winner et al.
<cit>  observed penetration of m. gallisepticum in hela- <dig> and cef cells occurred as early as five minutes after infection, and the intracellular mycoplasmas increased after  <dig> hours.
ureaplasmas have not been previously reported as cell invaders and have never been compared in their invasion rate.
in the present study, u. diversum showed a hasty invasion in hep- <dig> cells.
mollicute reference strains and the clinical isolates showed that these bacteria may have differences in growth and behavior when inoculated in animals or cell cultures  <cit> .
the high passage strains have been described as more adapted to axenic growth in contrast to the low passage clinical isolates that have shown to be more aggressive in experimental infections  <cit> .
even in erythrocytes, hela- <dig> and cef cells m. gallisepticum r low strain exhibited the highest invasion frequencies than the high passage strain  <cit> .
the authors suggested a loss or switching off of the genetic information in this species for the invasion process in the high passage strains.
in the present study, we observed the same frequency of invasion in the gentamicin invasion assay for the high and low passages clinical strains.
even when a field isolates, the higher passage ureaplasma may not lose or change yet the genetic expression for the studied invasion.
in fact these mollicutes are few studied and quite different, therefore, they may reveal additional features for these bacteria.
buim  observed that the high  and low passage isolates  of m. synoviae also showed similar adhesion and invasion into hep- <dig> cells and similarly surrounded the nucleus.
ueno et al.
<cit>  observed the same results with high and low passages of m. genitallium infecting hela and endometrial human cells.
in this study, both ureaplasma reference strains and clinical isolates were detected inside the cells similarly surrounding the perinuclear regions but not inside the nucleus.
the perinuclear arrangement was observed in other mollicutes  <cit> .
nevertheless, ueno et al.
<cit>  detected m. genitalium inside the nucleus after  <dig> minutes infection.
meseguer et al.
<cit>  observed abnormal fluorescence in nuclear images in infected cultures, but failed to confirm the location of m. pneumonie.
the invasion of mollicutes is not completely established and different mechanisms have been proposed based on the studied mollicute and infected cells.
yavlovich et al.
<cit>  showed the dependence of plasminogen-pg in the invasion process of m. fermentans mf.
the pg treated mf were able to invade hela cells in three hours, but not the untreated mf.
the phospholipase c  is detected in many walled bacteria and is considered a virulence factor for tissue damage.
in some mollicutes, plc was detected  <cit>  and associated with the cell invasion due to membrane and cytoskeleton modification.
the mycoplasmal plc was also associated with a host cell signal transduction cascade and the rearrangement of host cytoskeletal components  <cit> .
the invading mycoplasmas generate uptake signals that trigger the assembly of highly organized cytoskeletal structures in the host cells.
the invasion of m. penetrans is associated with tyrosine phosphorylation of a 145-kda host cell protein that activate plc to generate two additional messengers: phosphatidylinositol metabolites and diacylglycerol  <cit> .
these observations support the hypothesis that m. penetrans use phospholipase to cleave membrane phospholipids, thereby initiating the signal transduction cascade.
moreover, the plc appears to play a role in the escape from the primary vacuole and in gaining access to the cytoplasm  <cit> .
listeria monocytogenes deficient in plc are 500-fold less virulent in mice  <cit> .
the studied ureaplasma showed a high plc activity, without differences between the reference strains and the clinical isolates.
this activity explains similar behavior in hep- <dig> cells and suggests the role of plc as a factor for invasion of ureaplasma.
CONCLUSIONS
the biological consequences of mycoplasma invasion are not established.
mycoplasma infections are quite variable and the diversity of species and hosts potentiate the complexity of this context.
the intracellular location for these bacteria appears to be a comfortable niche for growth, allowing them to be more aggressive and more protected against immune response and antibiotics.
although u. diversum is a little studied species, its intracellular location adds this important feature to the understanding of mollicutes and explains their importance in bovine diseases.

 in mycoplasma and cell cultures were first associated in laboratory contamination. this contamination affects research by invalidating results in diagnosis. however interference by these bacteria in mammalian non phagocytic cell cultures has been used to study mollicute biology. the opportunism of mollicutes is a challenging subject. these microbes are diverse enough to explain their relationship variety with the host cells. the adhesion seems crucial for their pathogenicity. in addition, some mollicutes have been detected inside non naturally phagocytic cells. in fact, the intracellular location is well protected from the immune system and some antibiotics. the use of non-phagocytic cells to study mollicutes has been of great interest mainly since mycoplasma fermentans was initially considered a cofactor in the pathogenesis of aids. other mycoplasmas showed this same characteristic when inoculated in non-phagocytic cells such as m. fermentans, m. pneumoniae, m. genitalium and m. gallisepticum. ureaplasma diversum is a bovine-originated mollicute, first isolated in and considered a non-pathogenic species. although detected in healthy animals, it is currently considered a pathogenic species due to its strong association with cattle diseases such as placentitis, fetal alveolitis, abortion and birth of weak calves. as with most animal mycoplasmosis, the cause of ureaplasma-associated reproductive disease is multifactorial. in bulls, this ureaplasma is an important pathogen of the genital tract, involved in such diseases as lowered sperm motility, seminal vesiculitis, and epididymitis. nevertheless, little is known about the virulence and pathogenic mechanisms of this mollicute. because the invasion of u. diversum in not known, we inoculated this mollicute in hep- cells and observed this infection through confocal laser scanning microscopy and used a gentamicin invasion assay.  u. diversum adhesion and invasion on hep- cells observed by clsm the images of infected cells were from the apical surface to the basolateral region and differentiated the actin filaments in green, from the blue luminescence of nuclei. therefore the ureaplasmas were detected in red luminescence, discriminating their arrangements in the serial sections of the infected cells. the dil solution did not show ureaplasmal cytotoxicity and allowed for differentiating the hep- cells from ureaplasmal arrangements. non-infected hep- cells did not exhibit distinct intracellular dil fluorescence. the images obtained showed adhesion and invasion of u. diversum in hep- cells. after one minute of infection, a few ureaplasmal cells were detected scattered and inside the hep- cells. after minutes of infection, the ureaplasma persists, scattered, but increased inside the hep- cells. at and hours of infection, the microorganisms were detected mostly surrounding the perinuclear regions. the studied microorganisms showed no differences in their distribution when adhered to or inside the cytoplasm after hours of infection. ureaplasmal infection produced no cytopathic effects in hep- cells in the studied period. disposal of u. diversum in the infected hep- cells gentamicin invasion assay the ub medium promoted the growth of studied ureaplasmas. the exposure of inoculum size of ureaplasmas used for gentamicin allowed no recovery in ub medium. however the ureaplasma of infected hep- cells incubated with gentamicin and trypsinized allowed recovery of this microorganism. in this assay, it was possible to determine that the clinical isolates of ureaplasma revealed to be more concentrated in hep- cells than reference strains. this quantification was determined by 10-fold dilutions of ureaplasma obtained after gentamicin assay in ub medium and expressed as changing color units/ml. therefore, the internalization of studied ureaplasma in hep- was confirmed and quantified in this assay. gentamycin is impermeable to mammalian cells in the concentration used: it kills only the extra cellular ureaplasma but not the internalized bacteria. the rates of invasion were expressed as the percentage of ccu obtained after antibiotic exposure relative to the initial inoculum. the calculated p-value < e- test for equality of proportions with continuity correction, r project, vienna, austria allow for concluding that approximately 1% of the initial inoculum had survived the gentamicin treatment in type-strains and about 10% in clinical isolates. the atcc strain has a high passage in ub medium. no differences were observed in frequency of invasion between high and low passages clinical isolates. phospholipase c activity the ureaplasmas were initially cultured at 37°c for hours in one ml of ub broth with pnppc. the supernatants were evaluated at a wavelength of nm in a multiskan microplate reader. the phospholipase c activity was found in the studied ureaplasma and all produced high levels of this enzyme. the average activity was to pnppc hydrolysis. this was the highest level that allowed detection of this compound in the present study. the phospholipase c activity also measured in sonicated ureaplasmas cells. the average activity was to pnppc hydrolysis. these results showed that most activity is related to secreted enzyme. no differences were detected between the reference strains and clinical isolates. however this activity could be associated with a feature for invasion of ureaplasma. discussion adhesion and invasion has been studied in a few mollicutes, most being human-originated species. adhesion is considered an important feature to pathogenesis of these bacteria, and the invasion, a subsequent event, has been described in phagocytic or non phagocytic cells. therefore chronic and recurrent mycoplasmosis may be explained in part by the reported failures of antibiotic treatments and immune response escape. vancini & benchimol reported m. hominis invasion in trichomonas vaginalis and escaped from the vacuolization of trichomonad cytosol. this finding adds to understanding the challenging features of mollicute biology and their transmission among the hosts. consistent with other studied mollicutes, the infection described herein with u. diversum in hep- cells allowed for identifying this ureaplasma as another mammalian cell invader and may also explain and support prior findings on some ureaplasmal infections in bovines. cslm has been used to detect mollicute invasion in non phogocytic cells confirming its advantage in detecting u. diversum invasion. the gentamicin invasion assay also confirmed this finding. u. diversum was detected in hep- cells one minute after infection. m. penetrans has been observed as early as minutes after infection in hela cells, while in hep- cells, the invasion occurred after hours of infection. cell internalization after minutes was also detected for m. genitalium in hela cells, and the mycoplasmas remained inside the cells for days. winner et al. observed penetration of m. gallisepticum in hela- and cef cells occurred as early as five minutes after infection, and the intracellular mycoplasmas increased after hours. ureaplasmas have not been previously reported as cell invaders and have never been compared in their invasion rate. in the present study, u. diversum showed a hasty invasion in hep- cells. mollicute reference strains and the clinical isolates showed that these bacteria may have differences in growth and behavior when inoculated in animals or cell cultures. the high passage strains have been described as more adapted to axenic growth in contrast to the low passage clinical isolates that have shown to be more aggressive in experimental infections. even in erythrocytes, hela- and cef cells m. gallisepticum r low strain exhibited the highest invasion frequencies than the high passage strain. the authors suggested a loss or switching off of the genetic information in this species for the invasion process in the high passage strains. in the present study, we observed the same frequency of invasion in the gentamicin invasion assay for the high and low passages clinical strains. even when a field isolates, the higher passage ureaplasma may not lose or change yet the genetic expression for the studied invasion. in fact these mollicutes are few studied and quite different, therefore, they may reveal additional features for these bacteria. buim observed that the high and low passage isolates of m. synoviae also showed similar adhesion and invasion into hep- cells and similarly surrounded the nucleus. ueno et al. observed the same results with high and low passages of m. genitallium infecting hela and endometrial human cells. in this study, both ureaplasma reference strains and clinical isolates were detected inside the cells similarly surrounding the perinuclear regions but not inside the nucleus. the perinuclear arrangement was observed in other mollicutes. nevertheless, ueno et al. detected m. genitalium inside the nucleus after minutes infection. meseguer et al. observed abnormal fluorescence in nuclear images in infected cultures, but failed to confirm the location of m. pneumonie. the invasion of mollicutes is not completely established and different mechanisms have been proposed based on the studied mollicute and infected cells. yavlovich et al. showed the dependence of plasminogen-pg in the invasion process of m. fermentans mf. the pg treated mf were able to invade hela cells in three hours, but not the untreated mf. the phospholipase c is detected in many walled bacteria and is considered a virulence factor for tissue damage. in some mollicutes, plc was detected and associated with the cell invasion due to membrane and cytoskeleton modification. the mycoplasmal plc was also associated with a host cell signal transduction cascade and the rearrangement of host cytoskeletal components. the invading mycoplasmas generate uptake signals that trigger the assembly of highly organized cytoskeletal structures in the host cells. the invasion of m. penetrans is associated with tyrosine phosphorylation of a 145-kda host cell protein that activate plc to generate two additional messengers: phosphatidylinositol metabolites and diacylglycerol. these observations support the hypothesis that m. penetrans use phospholipase to cleave membrane phospholipids, thereby initiating the signal transduction cascade. moreover, the plc appears to play a role in the escape from the primary vacuole and in gaining access to the cytoplasm. listeria monocytogenes deficient in plc are 500-fold less virulent in mice. the studied ureaplasma showed a high plc activity, without differences between the reference strains and the clinical isolates. this activity explains similar behavior in hep- cells and suggests the role of plc as a factor for invasion of ureaplasma.  the biological consequences of mycoplasma invasion are not established. mycoplasma infections are quite variable and the diversity of species and hosts potentiate the complexity of this context. the intracellular location for these bacteria appears to be a comfortable niche for growth, allowing them to be more aggressive and more protected against immune response and antibiotics. although u. diversum is a little studied species, its intracellular location adds this important feature to the understanding of mollicutes and explains their importance in bovine diseases. 
the invasion in hep- cells of four clinical isolates and two reference strains of their ureaplasma was studied by confocal laser scanning microscopy and gentamicin invasion assay. the gentamicin invasion assay detected that 1% of the atcc strains were inside the infected hep- cells in contrast to 10% to the clinical isolates. the results presented herein will help better understand u. diversum infections, aswell as cellular attachment and virulence. understanding mollicutes is challenging due to their variety and relationship with host cells. a high level of phospholipase c activity was also detected in all studied ureaplasma.
 understanding mollicutes is challenging due to their variety and relationship with host cells. invasion has explained issues related to their opportunistic role. few studies have been done on the ureaplasma diversum mollicute, which is detected in healthy or diseased bovine. the invasion in hep- cells of four clinical isolates and two reference strains of their ureaplasma was studied by confocal laser scanning microscopy and gentamicin invasion assay.  the isolates and strains used were detected inside the cells after infection of one minute without difference in the arrangement for adhesion and invasion. the adhesion was scattered throughout the cells, and after three hours, the invasion of the ureaplasmas surrounded the nuclear region but were not observed inside the nuclei. the gentamicin invasion assay detected that 1% of the atcc strains were inside the infected hep- cells in contrast to 10% to the clinical isolates. a high level of phospholipase c activity was also detected in all studied ureaplasma.  the results presented herein will help better understand u. diversum infections, aswell as cellular attachment and virulence. 