Soil microorganisms control plant ectoparasitic nematodes in natural coastal foredunes Belowground herbivores can exert important controls on the composition of natural plant communities . Until now , relatively few studies have investigated which factors may control the abundance of belowground herbivores . In Dutch coastal foredunes , the root-feeding nematode Tylenchorhynchus ventralis is capable of reducing the performance of the dominant grass Ammophila arenaria -LRB- Marram grass -RRB- . However , field surveys show that populations of this nematode usually are controlled to nondamaging densities , but the control mechanism is unknown . In the present study , we first established that T. ventralis populations are top-down controlled by soil biota . Then , selective removal of soil fauna suggested that soil microorganisms play an important role in controlling T. ventralis . This result was confirmed by an experiment where selective inoculation of microarthropods , nematodes and microbes together with T. ventralis into sterilized dune soil resulted in nematode control when microbes were present . Adding nematodes had some effect , whereas microarthropods did not have a significant effect on T. ventralis . Our results have important implications for the appreciation of herbivore controls in natural soils . Soil food web models assume that herbivorous nematodes are controlled by predaceous invertebrates , whereas many biological control studies focus on managing nematode abundance by soil microorganisms . We propose that soil microorganisms play a more important role than do carnivorous soil invertebrates in the top-down control of herbivorous ectoparasitic nematodes in natural ecosystems . This is opposite to many studies on factors controlling root-feeding insects , which are supposed to be controlled by carnivorous invertebrates , parasitoids , or entomopathogenic nematodes . Our conclusion is that the ectoparasitic nematode T. ventralis is potentially able to limit productivity of the dune grass A. arenaria but that soil organisms , mostly microorganisms , usually prevent the development of growth-reducing population densities . Introduction Root herbivores play an important role in shaping the composition of natural plant communities -LRB- Brown and Gange 1990 -RRB- . Nematodes and insects represent the vast majority of the belowground herbivores -LRB- Brown and Gange 1990 ; Stanton 1988 -RRB- . Nematodes are more abundant than soil insects , and in some grassland ecosystems , nematodes are the dominant herbivores -LRB- Ingham and Detling 1986 -RRB- . Root-feeding nematodes have been estimated to take up as much as one quarter of the net primary production of grassland vegetation -LRB- Stanton 1988 -RRB- , and they affect plant quality -LRB- Davis et al. 1994 ; Troelstra et al. 2001 -RRB- , plant diversity , and vegetation succession -LRB- de Deyn et al. 2003 -RRB- . Root-feeding nematodes can also indirectly affect plant performance by their influence on bottom-up and top-down control of aboveground invertebrate herbivores -LRB- Bezemer et al. 2005 -RRB- . However , in spite of the increasing knowledge on the significant role of belowground herbivores in the control of plant abundance and plant community composition , relatively few studies have investigated which factors control the abundance of the belowground herbivores in natural ecosystems -LRB- Strong et al. 1996 , 1999 -RRB- . Herbivore abundance can be influenced by natural enemies -LRB- top-down -RRB- , by the host plant -LRB- bottom-up -RRB- , and by competition with other herbivores -LRB- horizontal control -RRB- . In -LRB- semi -RRB- natural ecosystems , most studies on the control of root-feeding nematodes have focused on plant quality -LRB- Yeates 1987 -RRB- , interspecific competition -LRB- Brinkman et al. 2004 , 2005 -RRB- , plant community composition -LRB- de Deyn et al. 2004 -RRB- , plant succession and soil conditions -LRB- Verschoor et al. 2002 -RRB- , and mycorrhizal fungi -LRB- de la Peña et al. 2006 -RRB- . Soil food web models assume root-feeding nematodes to be controlled by carnivorous nematodes and microarthropods -LRB- Hunt et al. 1987 ; Neutel et al. 2002 -RRB- . However , most biological control studies in agricultural systems focus on managing nematode abundance by parasitic soil microorganisms -LRB- Kerry 2000 ; Sikora 1992 -RRB- or mycorrhizal fungi -LRB- Hol and Cook 2005 -RRB- , suggesting that root-feeding nematodes are mainly controlled by microorganisms . Therefore , previous studies show little agreement and do not clearly predict how root-feeding nematodes will be controlled in natural ecosystems . Empirical data for top-down mechanisms are rare for terrestrial ecosystems relative to the many studies in aquatic systems -LRB- Walker and Jones 2001 -RRB- . In general , trophic cascades have been argued to be less common on land than in water -LRB- Polis and Strong 1996 -RRB- . Nevertheless , there is empirical evidence supporting the existence of trophic cascades in terrestrial plant -- predator -- prey systems -LRB- Schmitz et al. 2004 -RRB- . Tritrophic systems of plants , aboveground insect herbivores , and their natural aboveground enemies are the best-studied terrestrial examples of top-down and bottom-up herbivore controls -LRB- Carson and Root 1999 ; Rosenheim 1998 -RRB- . Below ground , tritrophic interactions may not essentially differ from what is known above ground -LRB- Bezemer and van Dam 2005 -RRB- , although rates of dispersal of organisms and chemical compounds will be lower than is mostly the case above ground -LRB- Rasmann et al. 2005 ; van der Putten 2003 -RRB- . Therefore , the challenge is , similar to that above ground -LRB- Schmitz et al. 2004 -RRB- , to assess what controls the abundance of root herbivores . This knowledge will enhance our understanding of belowground multitrophic interactions and their influences on plant performance and plant community composition . In the present study , the role of microarthropods , nematodes , and microorganisms in controlling the abundance of the root-feeding nematode Tylenchorhynchus ventralis -LRB- Loof 1963 -RRB- Fortuner and Luc -LRB- synonym Telotylenchus ventralis -RRB- was experimentally compared . This nematode is a polyphagous ectoparasite , which means that it is a quite generalistic root feeder that penetrates outer cortical cells with its stylet to collect and ingest cell contents -LRB- Yeates et al. 1993 -RRB- . T. ventralis is a root parasite of the dominant coastal foredune grass Ammophila arenaria -LRB- Marram grass -RRB- . In field soil , T. ventralis reaches densities that are 80 times lower than achieved when inoculated into sterilized dune soil -LRB- de Rooij van der Goes 1995 -RRB- . Whereas T. ventralis can strongly reduce growth of A. arenaria in sterilized soil , field densities in nonsterilized soil are too low to directly influence plant performance -LRB- de Rooij van der Goes 1995 -RRB- . The roots of A. arenaria are parasitized by an array of herbivorous nematodes ranging from ectoparasites to sedentary endoparasites -LRB- de Rooij van der Goes et al. 1995 -RRB- . The control mechanisms of root herbivorous nematodes in dunes appear to highly depend on the feeding type of the nematode , and even on the species of nematode . Whereas the sedentary root knot nematode Meloidogyne maritima -LRB- Jepson 1987 -RRB- Karssen , van Aelst and Cook is controlled by competition -LRB- Brinkman et al. 2005 -RRB- , the sedentary cyst nematode Heterodera arenaria -LRB- Cooper 1955 -RRB- Robinson , Stone , Hooper and Rowe appears to be controlled by bottom-up processes -LRB- van der Stoel et al. 2006 -RRB- . The migratory endoparasitic root lesion nematode Pratylenchus penetrans -LRB- Cobb 1917 -RRB- is controlled by arbuscular mycorrhizal fungi -LRB- de la Peña et al. 2006 -RRB- . Thus far , the factors that control the ectoparasitic nematode T. ventralis associated with A. arenaria are unknown . Previous studies showed bottom-up control of A. arenaria to occur only when the plants were severely growth reduced -LRB- de Rooij van der Goes et al. 1995 -RRB- . Alternatively , competition with cyst and root lesion nematodes is a potential factor controlling ectoparasitic nematodes -LRB- Eisenback 1993 -RRB- . However , endoparasitic nematodes did not control abundance of T. ventralis -LRB- Brinkman et al. 2004 -RRB- . In the present study , the top-down factors that may be involved in the control of T. ventralis populations were investigated in order to determine how belowground trophic interactions might influence plant performance and vegetation composition . To assess the top-down control of T. ventralis , three experiments were performed . The aim of experiment 1 was to elucidate the potential top-down control of T. ventralis by the dune soil community . In experiment 2 , the particular role of microorganisms was investigated by selective elimination of soil fauna -LRB- nematodes and microarthropods -RRB- . In experiment 3 , the hypothesis that emerged from experiment 2 , that soil microorganisms are the main cause of top-down control of T. ventralis , was tested . Here , we applied Koch 's postulates by collecting microorganisms , nematodes , and microarthropods from dune soil and adding them to sterilized soil inoculated with T. ventralis . New evidence that top-down control by soil microorganisms is the most important factor controlling the abundance of ectoparasitic nematodes in dune soil is presented and discussed . Materials and methods Soil In summer 2003 , soil samples were collected from mobile and stable foredunes at Voorne , The Netherlands -LRB- Latitude 51 ° 55 ′ N to Longitude 04 ° 05 ′ E -RRB- . The samples were collected along six transects parallel to the beach and 50 m apart . At each sampling station in the mobile and stable dune , 60 kg of soil was collected from the youngest root zone of A. arenaria . The soil was sieved -LRB- 0.5-cm mesh size -RRB- to remove plant parts and debris and stored in plastic bags at 4 °C until used -LRB- van der Stoel et al. 2002 -RRB- . Plants Seeds of A. arenaria were collected from the same foredune area and stored dry until used . In order to obtain seedlings , the seeds were germinated for 2 weeks on moist glass beads in a climate room at a 16/8 h light/dark regime at a temperature of 25/15 °C , respectively . When the first leaf was 2 -- 3 cm long , the seedlings were transplanted to 1.5-l plastic pots filled with 1,500 g of dune soil . In each pot , four seedlings of A. arenaria were planted , and the soil surface was covered with aluminum foil to protect the soil from desiccation . The soil moisture was adjusted to 10 % w/w and maintained at this level throughout the experiment by weighing the pots twice a week and resetting their initial weight using demineralized water . Once a week , full-strength Hoagland nutrient solution was added at a weekly rate of 12.5 ml pot − 1 for the first 3 weeks and then 25 ml pot − 1 , subsequently -LRB- Brinkman et al. 2004 -RRB- . This nutrient supply rate was effective to compensate for effects of nutrient release as a result of soil sterilization in dune soil -LRB- Troelstra et al. 2001 ; van der Putten et al. 1988 -RRB- . The experiments were carried out in a greenhouse at a day temperature of 21 °C ± 2 °C -LRB- day length 16 h -RRB- with additional light -LRB- to maintain a minimum of 225 μmol m − 2 s − 1 PAR with SON-T Agro lamps -RRB- and a night temperature of 16 °C . These temperatures are comparable with summer conditions in the field and are optimal for both plant and nematode development -LRB- Troelstra and Wagenaar unpublished results -RRB- . Experiments Experiment 1 : multiplication of T. ventralis in sterilized and nonsterilized dune soil In this experiment , the effect of soil origin -LRB- mobile and stable dunes -RRB- and soil organisms on multiplication of the ectoparasitic nematode T. ventralis was tested . Half of the soil was sterilized by gamma irradiation at an average dose of 25 kGray , which eliminates microorganisms and nematodes effectively from dune soil -LRB- de Rooij van der Goes et al. 1998 -RRB- . One week after the seedlings of A. arenaria had been transplanted , half the pots were inoculated with 50 T. ventralis pot − 1 . The noninoculated pots served as controls for effects of T. ventralis on plant biomass production . There were six replicates of each treatment . Experiment 2 : reproduction of T. ventralis in partially sterilized soil Multiplication of T. ventralis was studied in soils from which microarthropods and nematodes had been selectively removed by stirring the soil for 15 min at 1,500 rpm . This method has proven to effectively kill the soil fauna -LRB- de Rooij van der Goes et al. 1998 -RRB- . We confirmed this by inspecting the soil following stirring and found no live nematodes or microarthropods . The experiment was carried out as described above , but now the soil was completely sterilized by gamma irradiation -LRB- average 25 kGray -RRB- , partially sterilized by stirring to remove the soil fauna , or nonstirred in order to have a nonsterilized control soil . Each soil was inoculated with 0 , 25 , or 250 T. ventralis pot − 1 in order to examine any interaction between the effect of type of soil sterilization and nematode inoculation density . There were six replicates of each treatment . Experiment 3 : reinoculation of microorganisms , nematodes , and microarthropods into sterilized soil with T. ventralis In order to completely apply Koch 's postulates , microarthropods , nematodes , and microorganisms were extracted from the soil of mobile and stable coastal foredunes and inoculated alone and in all factorial combinations into sterilized dune soil . Then , seedling plants of A. arenaria were grown as in the previous experiment , and every pot was inoculated with 50 T. ventralis . All treatments were carried out in six replicates . The microorganisms were obtained by shaking soil samples of 100 g with demineralized water -LRB- 1:1 w/w -RRB- for 10 min and filtering the supernatant through a 20-μm mesh -LRB- Klironomos 2002 -RRB- . Prepared microbial filtrate contained no nematodes , but bacteria and fungi had readily passed through the filter . The pots with microorganisms were inoculated with 10 ml of the filtrate , which was 1/15 of the original soil density . For each pot , nematodes had been extracted from 1,500 g of nonsterile soil by Cobb 's method -LRB- Oostenbrink 1960 -RRB- and added in a suspension of 10 ml pot − 1 , so that nematode inoculation density corresponded with the density of nematodes in field soil . The nematode community added to the pots was analyzed microscopically -LRB- magnification 200 × -RRB- and consisted of plant parasites -LRB- T. ventralis , T. microphasmis , Pratylenchus spp , Paratylenchus spp. , Meloidogyne spp. , Rotylenchus spp. , Criconematidae -RRB- , bacterivores -LRB- Acrobeles spp. , Acrobeloides spp. , Chiloplacus spp. , Cephalobidae , Plectus spp. -RRB- , omnivores -LRB- Aporcelaimellus spp. , Microdorylaimus spp. -RRB- , and carnivores -LRB- Choanolaimus spp. -RRB- . Microarthropods were collected from nonsterile dune soil by wet sieving through 180-μm mesh and added as 10 ml of suspension pot − 1 , which corresponded with the field density of microarthropods . Demineralized water was added to all pots in equal amounts . Assessing the presence of microbial enemies on nematodes in field soil In order to confirm whether microbial enemies may occur on T. ventralis in the field , we extracted mobile nematodes from 100 cm3 of the field soil from each of the sampling sites using an adaptation of the Tray method -LRB- Whitehead and Hemming 1965 -RRB- . Half of the resulting nematode suspension was inspected using an inverted microscope -LRB- magnification 200 × -RRB- , and the nematodes were checked for symptoms of infection by bacteria or fungi . Nematodes infected by fungi were picked from the suspension and transferred to a corn-meal agar plate with antibiotics to encourage sporulation -LRB- Smith and Onions 1994 -RRB- , making possible identification of fungi that were previously found in a vegetative state . Identification of fungal natural enemies was done by observing mycelia and spore structure morphology and comparing this with the descriptions of Barron -LRB- 1977 -RRB- . Endospores of the parasitic bacterium Pasteuria spp. were recorded when observed attached to the nematode cuticle . Symptoms of infection by a nonlethal bacterial parasite Microbacterium nematophilum were assessed according to Sulston and Hodgkin -LRB- 1988 -RRB- . To detect whether nematode natural enemies may occur as dormant forms in the soil , nematode-baited sprinkle plates were used . Soil -LRB- 1 g -RRB- from each of the samples was sprinkled on water agar -LRB- 1 % -RRB- in a 9-cm-diameter Petri dish . A concentrated suspension of an estimated 500 Caenorhabditis elegans synchronized in the young adult stage -LRB- Sulston and Hodgkin 1988 -RRB- was added to the plates . A negative control containing nematodes only in water agar -LRB- 1 % -RRB- was used . The plates were sealed , kept at room temperature , and observed after 2 weeks and subsequently at weekly intervals up to 5 weeks -LRB- Barron 1977 -RRB- . Identification of fungal natural enemies was done as described above . Harvest All three experiments were harvested 12 weeks after inoculation of T. ventralis , allowing this nematode to complete two reproductive cycles -LRB- de Rooij van der Goes 1995 -RRB- . The nematodes were extracted from soil by Cobb 's decantation method and from the roots using a mistifier -LRB- Oostenbrink 1960 -RRB- . The numbers of T. ventralis were counted using a microscope -LRB- magnification 200 × -RRB- and expressed as numbers 100 g − 1 of dry soil . The roots and shoots of A. arenaria were dried for 48 h at 75 °C and weighed . Data analysis Normal distribution of data and homogeneity of variance were checked by inspection of the residuals after model fit -LRB- using the package Statistica 7 -RRB- . To obtain the normal distribution of data and homogeneity of variances , numbers of T. ventralis were log transformed in experiment 1 and square-root transformed in experiment 2 . In all three experiments , the soil origin -LRB- stable or mobile dune -RRB- did not affect significantly -LRB- P > 0.05 -RRB- the measured variables . Therefore , all data from treatments with those two soil origins was pooled , resulting in 12 replicates per treatment . Numbers of T. ventralis of experiment 1 were analyzed using one-way analysis of variance -LRB- ANOVA -RRB- with main the factor `` soil treatment . '' Two-way ANOVA with the main factors `` soil sterilization '' and `` nematode inoculation '' were performed for root and shoot biomass . Three-way ANOVA with the main factors `` stirring , '' `` sterilization , '' and `` inoculation density '' were performed for analyzing the numbers of T. ventralis shoot and root biomass in experiment 2 . Experiment 3 was analyzed by three-way ANOVA with the main factors `` invertebrates , '' `` nematodes , '' and `` microorganisms . '' Treatments were compared by posthoc analysis using Tukey honestly significant difference -LRB- HSD -RRB- tests -LRB- P < 0.05 -RRB- . Results Experiment 1 The numbers of T. ventralis at harvest differed significantly between sterilized and nonsterilized soils -LRB- F2 ,33 = 77.9 and P < 0.001 -RRB- . In the nonsterilized soil , addition of T. ventralis resulted in a significant increase of numbers at the end of the experiment compared with nonsterilized , noninoculated soil -LRB- Fig. 1 -RRB- . However , there were five times more T. ventralis in the inoculated sterilized soil than in the inoculated nonsterilized soil -LRB- Fig. 1 ; P < 0.05 ; sterilized soil without T. ventralis added was not included because the nematodes were absent -RRB- . These results show that multiplication of T. ventralis in nonsterilized soil was significantly enhanced by inoculation but that T. ventralis multiplication was significantly reduced by some factor in the nonsterilized soil that could be excluded by soil sterilization . Fig. 1Numbers of Tylenchorhynchus ventralis in 100 g of nonsterilized and sterilized dune soil 12 weeks after inoculation with T. ventralis . Error bars indicate standard error , and different letters above the bars indicate significant difference at P < 0.05 -LRB- experiment 1 -RRB- Soil sterilization influenced shoot biomass more than did T. ventralis inoculation -LRB- F1 ,44 = 117 , P < 0.001 for soil sterilization and F1 ,44 = 4.17 , P < 0.05 for inoculation , Fig. 2 -RRB- , and the effect of T. ventralis inoculation depended on soil sterilization -LRB- F1 ,44 = 7.06 , P < 0.05 -RRB- . Most shoot biomass was produced in sterilized soil , whereas T. ventralis inoculation significantly reduced shoot biomass -LRB- Fig. 2 -RRB- . As expected , the least shoot biomass was produced in nonsterile soil ; however , addition of T. ventralis caused no further reduction in growth -LRB- Fig. 2 -RRB- . As expected , root biomass was also strongly influenced by soil sterilization -LRB- F1 ,44 = 56.1 and P < 0.001 -RRB- , whereas the effect of T. ventralis addition was greater than for shoot biomass -LRB- F1 ,44 = 16.8 and P < 0.001 -RRB- . As for shoot biomass , the effect of T. ventralis inoculation on root biomass depended on soil sterilization -LRB- F1 ,44 = 9.87 and P < 0.005 -RRB- , which reflects that shoot biomass was significantly reduced by T. ventralis inoculation in the sterilized soil only -LRB- Fig. 2 -RRB- . Fig. 2Shoot and root biomass of Ammophila arenaria in sterilized and nonsterilized soil after 12 weeks from inoculation with Tylenchorhynchus ventralis . Error bars and letters above indicate significant differences at P < 0.05 -LRB- experiment 1 -RRB- Experiment 2 Significantly greater populations of T. ventralis developed in sterilized than in nonsterlilized soil at both inoculation densities -LRB- Tables 1 and 2 -RRB- . At the low-inoculation density , the number of the nematodes in nonsterliized soil was 30 times less than in sterilized soil and 15 times less at the high-inoculation density . There was no significant effect of soil stirring on the numbers of T. ventralis , although there was a trend -LRB- P = 0.06 -RRB- that stirring reduced T. ventralis multiplication . Table 1Three-way analysis of variance -LRB- ANOVA -RRB- of the numbers of Tylenchorhynchus ventralis in nonsterilized and sterilized , and stirred and nonstirred dune soil at three inoculation rates -LRB- 0 , 25 , 250 pot − 1 -RRB- after 12 weeks from inoculation to Ammophila arenaria . The data has been square-root transformed to achieve normal error distributiondfFPStirring -LRB- 1 -RRB- 13.4810.06 Sterilization -LRB- 2 -RRB- 1137.41 < 0.001 Inoculation density -LRB- 3 -RRB- 295.55 < 0.0011 × 210.8580.361 × 321.2220.302 × 3243.94 < 0.0011 × 2 × 321.4990.23 Error125Table 2 Effects of soil sterilization on numbers of Tylenchorhynchus ventralis in 100 g of soil -LSB- ± 1 standard error -LRB- SE -RRB- -RSB- 12 weeks from inoculation . Letters indicate significant differences at P < 0.05 -LRB- experiment 2 -RRB- NonsterilizedSterilized0 T. ventralis added0 .77 ± 0.22 a0 .08 ± 0.03 a25 T. ventralis added12 .3 ± 2.62 b390 ± 73.03 c250 T. ventralis added69 .2 ± 16.76 b , c1 ,162 ± 203.7 d As expected , both soil stirring and sterilization influenced shoot biomass -LRB- Fig. 3 ; Table 3 -RRB- . Shoot biomass was greater in sterilized than in nonsterilized soil and in stirred than in nonstirred soil ; however , inoculation density of T. ventralis did not influence the shoot biomass -LRB- Fig. 3 -RRB- . Root biomass was affected by stirring , soil sterilization , and inoculation density of T. ventralis , whereas effects of inoculation density depended on soil stirring as well as on soil sterilization -LRB- Table 3 -RRB- . If no nematodes were inoculated to the pots , soil sterilization almost doubled the root biomass . However , if inoculated with 25 or 250 T. ventralis per pot , the roots in sterilized soil with T. ventralis did not produce more biomass than those in nonsterilized soil -LRB- Fig. 3 -RRB- . Root biomass was significantly increased by soil stirring when no or few -LRB- 25 pot − 1 -RRB- T. ventralis were added to the pots , but there was no increase in root weight at the high inoculation rate . Fig. 3Effects of soil stirring , soil sterilization , and addition of Tylenchorhynchus ventralis on shoot and root biomass of Ammophila arenaria . Error bars and letters above indicate significant differences at P < 0.05 -LRB- experiment 2 -RRB- Table 3Shoot and root biomass of Ammophila arenaria 12 weeks after inoculation with nematodesdfShoot biomassRoot biomassFPFPStirring -LRB- 1 -RRB- 114.13 < 0.0018.69 < 0.01 Sterilization -LRB- 2 -RRB- 130.49 < 0.000126.8 < 0.001 Inoculation density -LRB- 3 -RRB- 21.9980.13910.53 < 0.0011 × 213.2880.0721.910.1691 × 321.9160.1513.622 < 0.052 × 322.1450.1216.452 < 0.011 × 2 × 321.9210.1511.9110.152 Error132The results of a factorial analysis of variance -LRB- ANOVA -RRB- with factors `` stirring , '' `` sterilization , '' and `` inoculation density '' -LRB- experiment 2 -RRB- Experiment 3 The multiplication of T. ventralis numbers was significantly reduced by adding a mixture of soil nematodes ; however , the effect of adding microorganisms was far greater -LRB- Fig. 4 ; Table 4 -RRB- . If microorganisms were present alone or in combination with other soil organisms , the number of T. ventralis was always less than when microorganisms were absent . On average , adding a suspension of nematodes reduced final numbers of T. ventralis by 15 % , whereas adding microorganisms reduced numbers of T. ventralis by 55 % -LRB- Fig. 4 -RRB- . Therefore , the effect of adding nematodes on T. ventralis multiplication was substantially weaker than the effect of microorganisms . Microarthropods did not have a significant effect on the numbers of T. ventralis . Adding microarthropods , nematodes , and microorganisms did not influence shoot or root biomass -LRB- P > 0.05 ; data not shown -RRB- . Fig. 4The effects of mixed nematode inoculum and microorganisms on Tylenchorhynchus ventralis multiplication . Error bars and letters above indicate significant differences at P < 0.05 -LRB- experiment 3 -RRB- Table 4The numbers of Tylenchorhynchus ventralis after 12 weeks from inoculationDegrees of freedom -LRB- df -RRB- F valueP valueMicroarthropods -LRB- 1 -RRB- 10.00060.981 Nematodes -LRB- 2 -RRB- 15.167 < 0.05 Microorganisms -LRB- 3 -RRB- 187.84 < 0.00011 × 211.7360.1911 × 310.6180.4342 × 310.6720.4151 × 2 × 310.0470.828 Error88The results of a three-way analysis of variance -LRB- ANOVA -RRB- with factors `` microarthropods , '' `` nematodes , '' and `` microorganisms '' -LRB- experiment 3 -RRB- In the suspension of nematodes obtained from the pots to which microorganisms had been added , 47.3 % showed signs of infection by culturable microbial enemies -LRB- Table 5 -RRB- . The fungal parasite Catenaria spp. was found infecting 16 out of 110 T. ventralis inspected , and an unidentified fungus was detected inside 30 destroyed of 110 T. ventralis checked . Bacterial attachment to the cuticle was also observed for six nematodes , a Paenibacillus-like organism was found on four nematodes , and Pasteuria spp. on two out of 110 nematodes . Table 5Microbial enemies in or attached to Tylenchorhynchus ventralis in a suspension obtained from microorganism treatment pots in experiment 3Microbial enemy detectedFraction of affected nematodes -LRB- % -RRB- Unidentified assimilative hyphae27.3Paenibacillus-like3.6 Catenaria spp. 14.5 Pasteuria spp. 1.8 Total47 .3 -LRB- Healthy -RRB- 52.7 Total nematodes examined100 -LRB- n = 110 -RRB- Assessing the presence of microbial enemies on nematodes in field soil The fungal natural enemies Catenaria spp. , Harposporium spp. , and Myzocytium spp. were found infecting nematodes extracted by the Tray method -LRB- Whitehead and Hemming 1965 -RRB- . The bacterium P.penetrans was attached to root knot nematodes -LRB- M. maritima -RRB- . The fungal genera were also detected using nematode-baited sprinkle plates . Some of the nematodes on the plates had a swollen region behind the anus not observed in the original culture or in the negative control . This is a symptom of infection by a nonlethal bacterial parasite , M. nematophilum . An unidentified trapping fungus with nonconstricting rings was also detected in the sprinkle plates but could not be identified , as it did not sporulate . These identifications may not have been exhaustive , but they confirmed the presence of antagonistic microorganism species on T. ventralis as well as in the soil from the field . Discussion In coastal foredunes , the root-feeding ectoparasitic nematode T. ventralis would significantly influence the pioneer grass A. arenaria if the density of this nematode was not controlled naturally . Our study strongly suggests that the natural control of T. ventralis in coastal foredune soil is mostly due to soil microorganisms . When inoculated into sterilized soil , numbers of T. ventralis were more than five times greater than when inoculated into nonsterilized soil , whereas selective elimination of soil fauna by stirring did not affect nematode numbers . These results from selective elimination studies were confirmed by isolating microarthropods , nematodes , and microbes and adding these together with T. ventralis to sterilized soil . Inoculation with soil microorganisms reduced T. ventralis more strongly than did inoculation with a nematode community consisting of other plant parasites , bacterivores , omnivores , and carnivores . The negative effect of the nematode community on T. ventralis density might have been due to competition with other root feeders . However , competition between T. ventralis and endoparasitic nematodes -LRB- i.e. H. arenaria , M. maritima and P. penetrans -RRB- occurred only if numbers of the competitors strongly exceeded present field densities -LRB- Brinkman et al. 2004 -RRB- . Therefore , the observed effect of adding nematodes on reducing T. ventralis was due to soil microorganisms cointroduced with the nematode suspension , carnivorous nematodes -LRB- Jairajpuri and Bilgrami 1990 -RRB- , or by effects of other non-plant-feeding nematodes . Our results highlight an important discrepancy in thinking about control mechanisms of plant-feeding -LRB- also called plant-parasitic -RRB- nematodes between biocontrol studies on the one hand and soil food web studies on the other . The majority of studies on the biocontrol of nematodes in agricultural ecosystems mostly focus on parasitic bacteria , such as P. penetrans and fungi -LRB- Kerry 2000 -RRB- , for example Arthrobotrys spp. and Pochonia spp. -LRB- Stirling and Smith 1998 -RRB- . Biological control practice usually does not consider microarthropods to be relevant for parasitic nematode control -LRB- Kerry and Gowen 1995 -RRB- , whereas the role of carnivorous nematodes has been considered -LRB- Mankau 1980 ; Yeates and Wardle 1996 -RRB- but not successfully used . According to the food web model used by Neutel et al. -LRB- 2002 -RRB- , root-feeding nematodes in coastal ecosystems are affected by predaceous mites and carnivorous nematodes . The role of microorganisms in nematode control is generally ignored in prominent soil food-web models -LRB- de Ruiter et al. 1993 ; Hunt et al. 1987 -RRB- . In our study system , however , soil microorganisms appeared to play a more important role than soil fauna in the control of plant ectoparasitic nematodes . In dune grasslands , the densities of soil fauna are usually rather low , perhaps too low to control significantly the abundance of nematodes -LRB- Petersen and Luxton 1982 -RRB- . Our results , therefore , support the view of biological control studies more than it supports soil food-web models for the control of the ectoparasitic nematode T. ventralis . Our results suggest that top-down control by natural enemies is more important for ectoparasitic feeding generalists , such as T. ventralis , than are competition -LRB- Brinkman et al. 2004 -RRB- or mycorrhizal fungi -LRB- Piśkiewicz and Hol unpublished results -RRB- , although these control mechanisms have been suggested for the other endoparasitic root-feeding nematode species in the same study system -LRB- Brinkman et al. 2005 ; de la Peña et al. 2006 ; van der Stoel et al. 2006 -RRB- . The control of T. ventralis in nonsterilized soil to which T. ventralis was added was not as good as in nonsterile soil , with only the background population of T. ventralis present . Earlier studies have shown that multiplication of T. ventralis is density and time dependent -LRB- de Rooij van der Goes 1995 -RRB- . Over time , a low inoculation density of nematodes may result in population increase , whereas a high inoculation density may result in population decline -LRB- de Rooij van der Goes 1995 -RRB- . However , we have no information as to how the microbial control shown in the present study may depend on nematode density , and subsequent long-term studies are required to further explore density dependence of nematode top-down control by microbes . The two screening methods used to detect microbial enemies of nematodes in the soil yielded a diversity of fungal and bacterial antagonists . The Tray method -LRB- Whitehead and Hemming 1965 -RRB- permitted the extraction of mobile stages of nematodes only , and therefore , dead or dying nematodes could not be screened . The nematode-baited sprinkle plate method , although useful for detecting microbial enemies in dormant forms in the soil , produced biased results that reflected the choice of nematode added , the bacterial-feeding nematode C. elegans . The fungal endoparasite Harposporium spp. infects nematodes that ingest its spores and therefore would not be able to infect plant parasites due to the narrow lumen of the stylet -LRB- Barron 1977 -RRB- . The bacterium M. nematophilum is thought to be a specialist parasite of C. elegans -LRB- Hodgkin et al. 2000 -RRB- . T. ventralis was attacked by a subset of the microbial enemy genera found in the soil , which reflects some specificity in the action of these microbes . Some microorganisms , namely Pasteuria spp. and the Paenibacillus-like organism , have not been detected in the dune soil , although they are widespread in the dune sites . This may be due to their absence or to presence in small numbers that are below the level of detection . The unidentified assimilative hyphae could be the result of attack by generalist trapping fungi . This was a first assessment to confirm the presence of nematode antagonistic microorganisms on T. ventralis , and further studies are needed to isolate culture and inoculate those antagonists in order to evaluate their contribution in root-feeding nematode control . Soil sterilization always led to increased root and shoot biomass . Previous studies have already shown that soil biota may reduce performance of A. arenaria -LRB- van der Putten et al. 1988 ; van der Stoel et al. 2002 -RRB- . The effect of soil sterilization was greater for root biomass than for shoot biomass . In sterilized soil , enhanced plant growth can be caused by nutrient release as a result of the soil sterilization process -LRB- Troelstra et al. 2001 -RRB- . We avoided different nutrient status of the sterilized and nonsterilized treatments by adding nutrient solution -LRB- van der Putten et al. 1988 -RRB- . When added to sterilized soil , the root-feeding nematode T. ventralis reduced root and shoot biomass of A. arenaria , and the effect of nematodes on root biomass increased with increasing inoculation density . Addition of T. ventralis to the nonsterile soil did not change root biomass , showing that the contribution of this nematode species to growth reduction of A. arenaria is limited -LRB- de Rooij van der Goes 1995 -RRB- . Our study shows that the dune soil not only contains biotic factors that reduce growth of A. arenaria but that there are also -LRB- micro -RRB- organisms that control population abundance of T. ventralis . In prairie grassland ecosystems , root-feeding nematodes have been assessed to account for reducing 58 % of aboveground biomass -LRB- Stanton 1988 -RRB- . These estimates are based on elimination trials using soil biocides . However , these studies did not verify the biocide effects by inoculation trials , and they also did not account for natural top-down control of the root-feeding nematodes . Our results suggest that when assessing the effects of root feeders on plant production , interactions of the root feeders with their natural predators needs to be taken into account as well . We conclude that soil microorganisms contribute to controlling the plant ectoparasitic nematode T. ventralis at a low population density in natural coastal foredunes . When not controlled , T. ventralis would be a key control factor for A. arenaria performance . Addition of other soil fauna , i.e. , nematodes and microarthropods , did not influence the abundance of T. ventralis as much as microorganism addition did , which confirms the marginal effects of soil fauna removal on reproduction of T. ventralis . Our results suggest that belowground multitrophic interactions can be crucial for plant performance . Revealing the precise identity of the microorganisms that have negative effects on the T. ventralis population , as well as mechanisms and involvement of the host plant , need further studies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

multitrophic interactions
ammophila arenaria
tylenchorhynchus ventralis


Progressive vertebral deformities despite unchanged bone mineral density in patients with sarcoidosis : a 4-year follow-up study Summary To evaluate the incidence of new and/or progressive vertebral deformities and changes in bone mineral density , we re-examined 66 patients with sarcoidosis after a follow-up period of four years . In 17 subjects -LRB- 26 % -RRB- new and/or progressive vertebral deformities were found , though BMD did not change significantly . Introduction Sarcoidosis is a T-cell driven chronic inflammatory disease . Although chronic inflammation has been associated with decreased bone mineral density as a result of the effects of cytokines on bone metabolism -LSB- 1 -- 4 -RSB- , we and others could not demonstrate changes in BMD in subjects with this condition , even if treated with glucocorticoids -LRB- GCs -RRB- . In a cross-sectional study of 124 subjects with sarcoidosis , BMD values similar to an age - and sex-matched reference population were found -LSB- 5 -RSB- . Comparable observations were made in three small studies in untreated patients -LSB- 6 -- 8 -RSB- . These studies also found a normal BMD relative to age and sex-matched controls , except for a small group of postmenopausal women in which BMD was moderately decreased at the spine in longstanding sarcoidosis only -LSB- 7 -RSB- . Although in our cross-sectional study normal BMD values were observed , increased levels of the bone resorption marker serum carboxy-terminal cross-linked telopeptide of type I collagen -LRB- ICTP -RRB- and the bone formation marker serum procollagen type I amino-terminal propeptide -LRB- PINP -RRB- suggestive of increased bone turnover were found -LSB- 5 -RSB- . ICTP levels correlated with markers of disease activity such as soluble IL-2 receptor -LRB- sIL2R -RRB- and angiotensin converting enzyme -LRB- ACE -RRB- . In addition , vertebral deformities suggestive of fracture were demonstrated in 20 % of the subjects studied in this series . This may imply that the fracture risk in sarcoidosis is increased due to an increased bone turnover with consequent changes in microarchitecture and decrease of bone strength which is not reflected by changes in BMD -LSB- 9 , 10 -RSB- . If so , this may result in progressive vertebral deformities during the course of the disease . For this reason we re-examined individuals with sarcoidosis four years after the initial measurements to determine the incidence of new and/or progressive vertebral deformities and their relation with changes in BMD . Subjects and methods Subjects Sixty-six of the 124 subjects with sarcoidosis that were studied in 2002 -LSB- 5 -RSB- agreed to participate in the follow-up study performed in 2006 . None of the 56 subjects who declined or were unable to participate had impaired mobility or a history of vertebral fractures . The mean age of this group was 45 years and did not differ with respect to gender or glucocorticoid -LRB- GC -RRB- use from the group of subjects that were re-examined in 2006 . Demographic , clinical and treatment data of the subjects studied in 2002 and 2006 are summarized in Table 1 . The group consisted of 22 pre-menopausal women , 11 post-menopausal women , and 33 men ; median age of the total group -LRB- all Caucasian -RRB- was 43 years -LRB- 20 -- 66 y -RRB- . The clinical records of all patients were reviewed . In 2002 patients were evaluated according to a standard protocol that included questionnaires , measurement of height and weight , lung function , measurement of BMD , a single energy densitometry of the spine , and laboratory evaluation -LSB- 5 -RSB- . In 2006 the same protocol was repeated . Informed consent was obtained from all participants and the study was approved by the medical ethics committee of our institution . Table 1Baseline and follow-up demographic , clinical , and treatment variables -LRB- n = 66 -RRB- VariableBaseline -LRB- n = 66 -RRB- Follow-up -LRB- n = 66 -RRB- P * Demographic variablesFemale sex33 -LRB- 50 % -RRB- Postmenopausal11 -LRB- 17 % -RRB- 14 -LRB- 21 % -RRB- nsAge , years43 -LRB- 20 -- 66 -RRB- Body mass index , kg/m226 .9 ± 5.727.2 ± 5.3 nsSmoking7 -LRB- 11 % -RRB- Daily dietary calcium intake , mg740 -LRB- 110 -- 2360 -RRB- 758 -LRB- 150 -- 1340 -RRB- nsClinical variablesDisease duration , years3 -LRB- 1 -- 22 -RRB- 7 -LRB- 5 -- 26 -RRB- Chest X-ray stage -LRB- 0-I-II-III-IV -RRB- 24/11/12 / 16/327/5 / 12/15/7nsFEV1 , % of predicted87 ± 2891 ± 26nsDLCO , % of predicted87 ± 1692 ± 18nsPhysical activity8 .6 ± 3.78.1 ± 3.7 nsLaboratory values -LRB- in serum -RRB- Calcium , mmol/l -LRB- 2.1 -- 2.6 -RRB- 2.4 ± 0.12.4 ± 0.08 ns1 ,25 -LRB- OH -RRB- 2D3 , nmol/l -LRB- 0.040 -- 0.200 -RRB- 0.14 ± 0.03 ACE , U/l -LRB- 9 -- 25 -RRB- 22.5 ± 9.815.3 ± 7.90.001sIL-2R , kU/l -LRB- 241 -- 846 -RRB- 654 -LRB- 188 -- 4315 -RRB- Hs-CRP , mg/l -LRB- < 10 -RRB- 3.2 -LRB- 0.2 -- 191 -RRB- 2.0 -LRB- 1 -- 16 -RRB- < 0.05Z-score ICTP0 .7 ± 1.4Z-score PINP − 0.1 ± 0.9 Treatment variablesGC use never31 -LRB- 47 -RRB- 26 -LRB- 39 -RRB- nsGC use previous14 -LRB- 21 -RRB- 25 -LRB- 38 -RRB- < 0.01 GC use current21 -LRB- 32 -RRB- 15 -LRB- 23 -RRB- nsLifetime GC dose , mg9240 -LRB- 200 -- 48750 -RRB- 11187 -LRB- 200 -- 56700 -RRB- < 0.001 Daily dose , mg12 .4 ± 6.210.5 ± 3.3 < 0.05 Started on bisphosphonates after baseline measurement6 -LRB- 9 -RRB- Clinical risk factors for osteoporosisFracture2 -LRB- 2/24 = 8 % -RRB- 5 -LRB- 5/28 = 18 % -RRB- nsLow body weight -LRB- < 60 kg -RRB- 8 -LRB- 12 -RRB- 7 -LRB- 11 -RRB- nsSevere immobilization00nsLow physical activity index ≤ 518 -LRB- 27 -RRB- 18 -LRB- 27 -RRB- nsMother with hip fracture6 -LRB- 9 -RRB- 7 -LRB- 11 -RRB- nsData are given as mean ± SD , median -LRB- range -RRB- or number -LRB- % -RRB- ; * = p value between baseline and follow-up measurement . Reference parameters in bracketsAbbreviations : GC , glucocorticoid ; FEV1 , forced expiratory volume in one second ; DLCO , diffusion capacity for carbon monoxide ; 1,25 -LRB- OH -RRB- 2D3 , 1,25 dihydroxyvitamin D ; ACE , angiotensin converting enzyme ; sIL-2R , soluble interleukin-2 receptor ; Hs-CRP , high-sensitivity C-reactive protein ; ICTP , carboxy-terminal cross-linked telopeptide of type I collagen ; PINP , procollagen type I amino-terminal propeptide Pulmonary evaluation Lung function measurements , including forced expiratory volume in one second -LRB- FEV1 -RRB- and forced vital capacity -LRB- FVC -RRB- , were measured with a pneumotachograph . The diffusion capacity for carbon monoxide -LRB- DLCO -RRB- was measured using the single-breath method -LRB- both Masterlab , Jaeger , Würzburg , Germany -RRB- . Values were expressed as a percentage of those predicted -LSB- 11 -RSB- . Chest radiographs were graded according to the radiographic staging of DeRemee -LRB- 0 to III -RRB- , adding stage IV , the end stage of lung fibrosis -LSB- 12 , 13 -RSB- . All interpretations were made by a radiologist who was blinded to the patient 's history . Laboratory assays Serum 1,25-dihydroxyvitamin D concentration was determined by radioimmuno-assay using a commercially available kit -LSB- -LRB- IDS Ltd , Boldon , England , interassay coefficient of variation -LRB- IE-CV -RRB- 18 % , intra-assay CV -LRB- IA-CV -RRB- 15 % -RRB- -RSB- . High-sensitivity C-reactive protein -LRB- hs-CRP -RRB- was measured by particle-enhanced immunonephelometry on the BN Prospec -LRB- Dade Behring -RRB- . The detection limit is 0.175 mg/L and the measuring range is 0.175 -- 1100 mg/L . Soluble IL-2 receptor -LRB- sIL-2R -RRB- was determined on the IMMULITE automated analyzer , by means of a two-site chemiluminescent enzyme immunometric assay with a measuring range of 50 -- 7500 kU/L -LRB- Diagnostic Product Corporation , Los Angeles , CA , cat no LKIP1 -RRB- . Serum angiotensin converting enzyme -LRB- ACE -RRB- was measured using a colorimetric method . The precision of the ACE assay was < 5.6 % and the reference interval for ACE was 9 -- 25 U/L . As a marker for bone formation , serum procollagen type I amino-terminal propeptide -LRB- PINP -RRB- was measured . As a marker for bone resorption , serum carboxy-terminal cross-linked telopeptide of type I collagen -LRB- ICTP -RRB- was assessed . Both PINP -LRB- IE-CV 3.2 % , IA-CV 2.5 % , lowest detectable concentration 0.4 μg/l -RRB- and ICTP -LRB- IE-CV 3.5 % , IA-CV 2.3 % , lowest detectable concentration < 0.1 μg/l -RRB- were measured using commercial RIA kits -LRB- Orion Diagnostica Oy , Espoo , Finland -RRB- . To adjust for age and gender Z-scores for these bone markers were obtained using a Dutch reference group -LRB- 300 women , 150 men -RRB- , checked for normal BMD of the lumbar spine and femur and normal 25-hydroxyvitamin-D levels -LSB- 14 , 15 -RSB- . Bone mineral density and vertebral morphometry Bone mineral density -LRB- BMD -RRB- was measured by dual X-ray absorptiometry -LRB- DXA , Hologic QDR 4500 -RRB- . In 2002 only the BMD of the hip was measured . In 2006 the BMD of both the hip and of the lumbar spine were determined . As reference group for the hip the NHANES III database -LRB- sex - and age-matched -RRB- was used . A standard protocol as described previously was used for measurement of BMD . To adjust for age and gender , Z-scores were used . To examine changes in Z-scores between baseline and follow-up measurements a Δ Z-score was calculated reflecting the difference between the Z-score at follow-up and the Z-score at baseline . Furthermore , after bone density measurement a lateral single energy densitometry of the thoracic and lumbar spine for vertebral fracture assessment -LRB- VFA -RRB- was performed -LRB- also called Morphometric X-ray absorptiometry -LRB- MXA -RRB- -RRB- -LSB- 16 -RSB- . The scans obtained were analyzed twice by one trained operator -LRB- intra-observer correlation : 0.85 -RRB- , using the semi-quantitative method of Genant -LSB- 17 -RSB- . In addition we measured every vertebra quantitatively . The observer was blinded to the T-score values and to the values of the first set of measurements . After visual examination six points were placed on each vertebral body from T4 to L4 . From these points three vertebral heights were measured anterior -LRB- Ha -RRB- , mid -LRB- Hm -RRB- and posterior -LRB- Hp -RRB- . On the basis of the average score of these morphometric measurements , ratios were calculated and a prevalent vertebral deformity was defined as a reduction of height of 20 % or more -LRB- Ha/Hp ; Hm/Hp and Hp/Hp below -RRB- . Severity of deformities was assessed using the scoring system of Genant -LSB- 17 -RSB- . A score of ` 0 ' was assigned to normal , non-fractured vertebra ; ` 1 ' for a mild deformity -LRB- 20 -- 25 % reduction in anterior , middle or posterior vertebral height -RRB- ; ` 2 ' for a moderate deformity -LRB- 25 -- 40 % reduction -RRB- and ` 3 ' for a severe deformity -LRB- > 40 % reduction -RRB- . A new vertebral deformity was scored if a normal vertebra -LRB- grade 0 -RRB- became deformed -LRB- grade ≥ 1 -RRB- and a progressive deformity if the grade increased -LSB- 17 -RSB- . Questionnaires Calcium intake of all patients was scored in 2002 as well as 2006 on the basis of a detailed dietary list . Known clinical risk factors for osteoporosis -LRB- weight below 60 kg , mother with hip fracture , history of fractures after age 50 , menopausal status and severe immobilization -RRB- as well as daily activities and exercise were assessed by a validated questionnaire -LSB- 18 -RSB- , in which sports , daily and work activities are scored with a minimum of zero and a maximum of eighteen . GC therapy was evaluated by means of a patient questionnaire and verified using all the records of the patient 's pharmacist . It was scored as never , previous or current use and if subjects were currently using GCs , the daily dose was noted . Statistics Student t-tests , chi-square tests , and one-way ANOVAs were used , depending on the variables and subgroups tested . Depending on the analysis , change scores or actual scores were used . Patients with new and/or progressive vertebral deformity were clustered for the multivariate and the receiver-operating characteristics -LRB- ROC -RRB- analysis . Multivariate logistic regression analyses was performed to assess the strength of association between the incidence of new and/or progressive vertebral deformities and gender , age , weight , clinical risk factors , GC use , lifetime GC dose , daily GC dose , disease activity , bone markers , calcium intake , physical activity and BMD measurements . The variables that were entered in the multivariable analysis were those variables that appeared related -LRB- p < 0.10 -RRB- to this outcome measure in univariate analyses . Odds ratio -LRB- OR -RRB- and its 95 % confidence intervals -LRB- CI -RRB- were calculated by using SPSS version 12.0 . ROC analysis was used to assess the ability of various levels of the T-score femoral neck to predict the incidence of a new and/or progressive vertebral deformity . The ROC curve indicates the probability of a true-positive result as a function of the probability of a false-positive result for all possible threshold values -LSB- 19 -RSB- . A p value < 0.05 was considered statistically significant . Results Bone mineral density and bone turnover parameters The results of BMD measurements are shown in Table 2 . The BMD of the total group remained unchanged after a median follow-up of 45 months -LRB- range 35 -- 49 months -RRB- . When stratifying patients according to GC use , no decrease in each of the subgroups was found . Patients that never used GCs showed a Δ Z-score of the femoral neck -LRB- FN -RRB- of 0.03 ± 0.36 and a Δ Z-score of the trochanter of − 0.08 ± 0.37 . In patients with previous use of GCs these Δ Z-scores were 0.10 ± 0.36 and 0.22 ± 0.43 , respectively . Even the group currently on GCs revealed no decrease of Z-score -LRB- Δ Z-score FN : 0.06 ± 0.30 and Δ Z-score trochanter : 0.00 ± 0.18 -RRB- and also the subgroup of postmenopausal women -LRB- n = 11 -RRB- did not show significant bone loss -LRB- Δ Z-score FN : 0.06 ± 0.45 and Δ Z-score trochanter : − 0.05 ± 0.52 -RRB- . In the total group , bone turnover parameters at baseline showed an increased Z-score of ICTP compared to norm scores -LRB- 0.7 , 95 % confidence interval -LRB- CI -RRB- : 0.4 -- 1.1 ; p < 0.001 -RRB- . on the other hand , the marker of bone formation -LRB- Z-score PINP -RRB- did not differ from the reference population . Table 2BMD variables at baseline and follow-up for the total group -LRB- n = 66 , median follow-up duration 45 months -LRB- range 35 -- 49 months -RRB- VariableBaseline measurementFollow-up measurementP * BMD , mean ± SD gm/cm2Femoral neck0 .84 ± 0.120.83 ± 0.12 nsTrochanter0 .74 ± 0.130.74 ± 0.12 nsTotal hip0 .97 ± 0.130.98 ± 0.14 nsLumbar spine1 .04 ± 0.14Z-score , mean ± SDFemoral neck0 .17 ± 1.00.23 ± 1.1 nsTrochanter0 .27 ± 1.10.32 ± 1.1 nsTotal hip0 .18 ± 1.00.32 ± 1.00.001 Lumbar spine0 .03 ± 1.4T-score , mean ± SDFemoral neck − 0.42 ± 1.0 − 0.46 ± 1.0 nsTrochanter − 0.02 ± 1.1 − 0.03 ± 1.0 nsTotal hip − 0.14 ± 1.0 − 0.10 ± 1.0 nsLumbar spine − 0.50 ± 1.3 Abbreviations : BMD , bone mineral densityP * = p value between baseline and follow-up measurement Clinical fractures and vertebral deformities Three new non-vertebral fractures occurred during the follow-up period . These included a hip fracture -LRB- twice in the same patient -RRB- , an ankle fracture and a fracture of the thumb . All these fractures were related to trauma and occurred in subjects older than 50 years . Morphometric data are summarized in Table 3 . In 2002 vertebral deformities -LRB- ratio of < 0.80 -RRB- were found in 19 vertebrae of 13 subjects . Seventeen of these were wedge and two biconcave deformities . No crush deformities were seen . The majority of these deformities were found in the low thoracic region . At follow-up a new vertebral deformity was scored if a normal vertebra -LRB- grade 0 -RRB- became deformed -LRB- grade ≥ 1 -RRB- and a progressive deformity if the grade increased -LSB- 17 -RSB- . With this method , 36 vertebral deformities were found in 21 subjects . In one subject a vertebral deformity -LRB- ratio 0.78 of T11 -RRB- found in 2002 was not found at follow-up -LRB- ratio 0.81 -RRB- . So , in total nine new subjects revealed one or more vertebral deformities , which means an increase of vertebral deformities from 20 to 32 % of the subjects studied -LRB- p < 0.05 -RRB- . From the 21 subjects with a vertebral deformity in 2006 , 17 subjects -LRB- 26 % of total group -RRB- were diagnosed with one or more new or progressive vertebral deformities and in four subjects the deformity was unchanged compared to baseline . Data on number and severity of the deformities can be found in Table 3 . Six patients were started on a bisphosphonates after baseline measurement and from these six patients , two had a new or progressive vertebral deformity at follow-up . Table 3Number and grade of deformitiesBaselineFollow-upNo . of subjects with deformity13 -LRB- 20 % -RRB- 21 -LRB- 32 % -RRB- * No . of deformities Mild1728 Moderate28 Severe00Total1936 * P < 0.05 between number of subjects with deformity at baseline and follow-up Comparing the groups with and without new or progressive vertebral deformities at follow-up , no differences in Δ Z-scores of BMD of the trochanter or femoral neck -LRB- FN -RRB- were found -LRB- Δ Z-score trochanter − 0.02 ± 0.41 and 0.08 ± 0.38 respectively and Δ Z-score FN 0.01 ± 0.32 and 0.08 ± 0.35 -RRB- . In addition no differences in baseline Z-scores of ICTP and PINP were seen between these groups . Multivariable logistic regression analysis , including factors that correlated in the univariate analysis , revealed that a T-score of the femoral neck at baseline -LRB- OR per 1 SD T-score reduction = 2.5 -LRB- CI : 1.0 -- 5.9 -RRB- , p = 0.04 -RRB- , and a mother with a hip fracture -LRB- OR = 14.1 -LRB- CI : 1.4 -- 142,6 -RRB- , p = 0.02 -RRB- were determinants of a new and/or progressive morphometric vertebral deformity at follow-up measurement . Factors such as age , gender , calcium in take , GC use , daily GC dose , lifetime GC dose , disease activity , bone markers , radiographic stage and disease duration at baseline did not predict new and/or progressive vertebral deformities . The threshold level of the T-score FN that maximized the combined specificity and sensitivity on the ROC curve -LRB- Fig. 1 -RRB- was < − 0.45 for predicting a new and/or progressive deformity -LRB- sensitivity 88 % , specificity 51 % -RRB- . Fig. 1ROC curve using Femoral neck T-score to identify patients with new and/or progressive vertebral deformity . Area under the curve -LRB- AUC -RRB- : 0.72 . Arrow : The threshold level of T-score FN that maximized combined specificity and sensitivity was < − 0.45 -LRB- sensitivity 88 % , specificity 51 % -RRB- Discussion In this cohort of subjects with sarcoidosis , a high prevalence of morphometric vertebral deformities suggestive of fracture was found , as well as a substantial increase in vertebral deformities during a follow-up period of four years . In 2002 20 % of subjects were diagnosed with vertebral deformities according to the criteria of Genant -LSB- 17 -RSB- , which increased to 32 % of all subjects in 2006 . In parallel , the total number of deformities in these subjects almost doubled . However , BMD of the trochanter and femoral neck did not change over time and BMD of the lumbar spine at follow-up measurement did not differ from the reference population . These data are suggestive of an increased risk of progressive vertebral deformities in individuals with sarcoidosis despite preservation of BMD . Although data on prevalent or incident fractures in younger healthy populations are lacking , data from other studies suggest that the incidence and prevalence of vertebral deformities in this population are indeed high . Prevalence rates of 30 % asymptomatic vertebral fractures are demonstrated in elderly post-menopausal women on chronic GC therapy using the same techniques -LSB- 20 -RSB- . In a previous study in 60 subjects -LRB- mean age 49 ± 13 years -RRB- with differentiated thyroid carcinoma we found vertebral deformities in 7 % of patients -LSB- 21 -RSB- . Data from the European Vertebral Osteoporosis Study -LRB- EVOS -RRB- , a very large cross-sectional population based study on European subjects aged 50 to 79 years , showed a prevalence of vertebral deformities of 12 % -LRB- range 6 -- 21 % -RRB- in males and females -LSB- 22 -RSB- . In the Rotterdam study , in which 3469 men and women aged 55 years and older were studied , the prevalence of vertebral deformity suggestive of fracture was 6.9 % in men and 7.5 % in women -LSB- 23 -RSB- . The epidemiology of vertebral fractures in women aged 50 -- 54 years turned out to vary in different countries from 4.7 % -- 11.5 % -LSB- 24 -RSB- . All these studies indicate that the fracture risk in subjects with sarcoidosis is substantial , regardless the differences in populations studied and differences in methodology . A new vertebral deformity was found in 15 subjects -LRB- 23 % -RRB- . To identify incident deformities several approaches can be followed . Measurement of changes in vertebral heights of the same vertebral body from a baseline to a later radiograph in which a decrease in height of 15 or 20 % or 4 mm is suggestive of fracture -LSB- 25 , 26 -RSB- , changes in indices of vertebral area -LSB- 27 -RSB- or changes in the number or presence of prevalent deformities -LSB- 17 , 28 -RSB- . Black and coworkers evaluated these different approaches and concluded that none of these were consistently better than any other method -LSB- 29 -RSB- . As we aimed to assess the change of numbers of subjects with one or more vertebral deformities over time we used the last method -LSB- 17 -RSB- , in which changes in number of prevalent deformities are scored . A comparable approach was followed in the European Prospective Osteoporosis Study -LRB- EPOS -RRB- -LSB- 30 -RSB- , which revealed an incidence of new deformities of 3.4 % after a similar follow-up period . As the mean age of subjects included in this study was substantially higher than that of our cohort , these data can not be used as a reference , although it is likely that in younger age groups even lower incident deformities would be observed . The high prevalence of vertebral deformities at baseline , the significant increase of more than 50 % of subjects after follow-up with one or more deformity and the increase of severity of prevalent deformities all imply that sarcoidosis is a relevant risk factor for vertebral deformity . What is the underlying mechanism of this predisposition to vertebral deformities in view of the lack of effects on BMD in sarcoidosis ? The load bearing capacity of bone , also referred to as ` whole bone strength ' , depends on the amount of bone , the spatial distribution of the bone mass , and the intrinsic properties of the materials that comprise the bone . Thus , properties at the cellular , matrix , micro - and macro-architectural levels may all impact the mechanical properties of bone -LSB- 31 , 32 -RSB- . Apparently , in sarcoidosis mechanisms are involved that influence bone strength without having a significant impact on bone mass . As we found in the total group an increased marker of bone resorption -LRB- Z-score ICTP -RRB- at baseline , one of the possible mechanisms could be increased bone remodelling with a negative effect on bone micro-architecture that is not reflected by a change in BMD . It is well known that chronic inflammatory diseases influence bone physiology by the production of cytokines stimulating bone turnover -LSB- 1 , 2 , 33 -RSB- . Increased bone remodelling is associated with an increased bone fragility and thus fracture risk -LSB- 34 , 35 -RSB- and in postmenopausal women the level of bone turnover turned out to be an as strong and independent predictor of fractures as BMD -LSB- 36 , 37 -RSB- . These data may support the hypothesis that the chronic inflammatory state in sarcoidosis results in increased bone remodelling with a negative effect on bone strength and thus an increased fracture risk . No changes in BMD in the group currently treated with GCs were found . This is unexpected as GCs are known to effect BMD via several mechanisms with consequent decrease of BMD . It may well be that this is due to intermittent GC use , as most of our patients were on intermittent glucocorticoids . Other studies have demonstrated that intermittent GC use has no major effects on BMD -LSB- 38 -RSB- . A recent large retrospective cohort study on clinical fracture risk among patients from the UK General Practice Research Database showed that intermittent use of high dose of oral GCs was associated with only a small increase in the risk of osteoporotic fractures -LSB- 39 -RSB- . Despite the on average normal BMD , we found T-score of the femoral neck and a family history of hip fractures to be predictors of a new and/or progressive vertebral deformity . This suggests that the combination of a lower BMD in combination with the increased bone turnover in sarcoidosis predisposes to progressive vertebral deformity . If so , this would mean that in these high risk individuals preventive treatment should be considered to reduce fracture risk . Controlled trials are needed , however , to substantiate this suggestion . One of the limitations of our study is the lack of an age and sex matched control population . Unfortunately data on vertebral deformities in younger populations are at present not available . The aim of this study was , however , not to compare sarcoidosis patients with healthy subjects , but to follow a cohort of these patients and to compare follow-up with baseline measurements . Another limitation is the use of morphometric X-ray absorptiometry -LRB- MXA -RRB- instead of spine radiographs . MXA is less reliable for the detection of deformities at the upper thoracic spine , where deformities are less frequent as compared to the lumbar and mid-thoracic spine . A recent study comparing MXA with lateral spine X-ray found that vertebral morphometry using MXA allowed diagnosis of vertebral fracture in the lumbar and mid thoracic spine , where vertebral fractures are most common -LSB- 40 -RSB- . The advantage of MXA is the low dose of radiation and the convenience of the technique for patients . The present quality of the images , with ongoing refinement of this technology , is considered sufficient to be used for the diagnosis of vertebral deformity consistent with fracture -LSB- 41 -RSB- . Furthermore there is a lack of a `` gold standard '' for VFA . We followed the method of Genant -LSB- 17 -RSB- , which is based on a reduction of the ratios of anterior , middle or posterior heights and all measurements were performed twice to improve accuracy . This is the simplest and most practical method -LSB- 42 -RSB- and an association with future fracture risk is documented -LSB- 43 , 44 -RSB- . The above mentioned EVOS study , however , applied the methodology described by McCloskey and Eastell and co-workers in which measurements are corrected for normal variations in vertebral shape -LSB- 25 -RSB- . Relative to the method of Genant , the method of Eastell -LSB- 25 -RSB- or McCloskey -LSB- 28 -RSB- may have resulted in lower prevalences of vertebral deformities . This does not , however , explain the differences in prevalence of vertebral deformities reported elsewhere and in this paper . The restrictions of the methodology are also the limited ability to provide a differential diagnosis for the detected deformities , a lower sensitivity for milder fractures and the inability to evaluate the uppermost thoracic levels . Other disorders that may cause changes in vertebral shape involve congenital abnormalities and conditions as severe osteoarthritis -LSB- 45 -RSB- and Scheuermann 's disease . We have , however , no indications that these relatively rare conditions may have interfered with our observations . In conclusion , we have shown that in subjects with sarcoidosis the number of vertebral deformities , diagnosed with morphometric assessment , increases during the course of this disease despite preservation of BMD . Although this is an uncontrolled study , it appears that subjects with sarcoidosis have an increased fracture risk , even if BMD is normal . High risk individuals can be identified by a low-normal BMD and by a family history of hip fractures . Probably these individuals will benefit from therapies that increase bone strength . A T-score FN below − 0.45 may be used to identify these individuals with a high sensitivity and an acceptable specificity . Studies evaluating the effects of such therapies in individuals with sarcoidosis are however clearly needed .

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O

bone mineral density
sarcoidosis
vertebral deformities