BACKGROUND
the rickettsial bacterium ehrlichia ruminantium is a causative agent of heartwater, the disease of ruminants transmitted by ticks of the genus amblyomma  <cit> .
heartwater is not only responsible for high economic losses in endemic countries  <cit> , but is also suggested to be a potential emerging zoonosis since the pcr and sequence detection of the pathogen's presence in three fatal human cases although the cytological examination and bacterial isolation were not achieved  <cit> .
the disease is established in nearly all countries of sub-saharan africa and some islands of the caribbean, from where it threatens the american mainland  <cit> .
in the usa, three ehrlichia species, namely e. canis, e. chaffeensis, and e. ewingii, are known to exist  <cit> .
recently, panola mountain  ehrlichia, which is closely related to e. ruminantium, was discovered as a novel zoonotic ehrlichia in the state of georgia  <cit> .
active surveillance using a reliable method which can discriminate e. ruminantium from these other ehrlichia species is an asset in preventing introduction of heartwater into the usa.
in heartwater endemic countries, conventional diagnosis is based upon clinical signs and microscopic examination of post-mortem brain smears.
as a more reliable and sensitive diagnostic method, several pcr-based assays have been developed for the detection of e. ruminantium, including conventional pcr  <cit> , nested pcr  <cit> , and real-time pcr  <cit> .
among them, the pcs <dig> real-time pcr taqman probe assay provides the best sensitivity with a detection limit of one gene copy per reaction, which is  <dig> times higher than that of conventional pcs <dig> pcr  <cit> .
however, this assay was reported to cross-react with both e. chaffeensis and e. canis  <cit> .
moreover, although this assay performs well in the sensitive detection and quantification of e. ruminantium, it is not readily transferable to low-technology settings where there is limited access to expensive fluorescence detector based thermocyclers.
loop-mediated isothermal amplification  assay is a rapid dna amplification method originally developed by notomi et al.
<cit> , and it has been applied for the detection of viral  <cit> , bacterial  <cit> , fungal  <cit> , and parasitic agents  <cit> , but it has never previously been applied to rickettsial agents.
the method requires a specially designed primer set that recognizes at least six independent regions of the target gene, which increases the specificity as well as the rapidity of the reaction.
lamp results are visualized by turbidity that can be seen by the naked eye  <cit> , and optionally by agarose gel electrophoresis or by addition of fluorescent dyes visualized under uv light  <cit> .
since the bst dna polymerase used in lamp allows strand displacement-dna synthesis, lamp reactions are performed under isothermal conditions using a simple incubator, such as a water bath or heating block.
furthermore, lamp reagents are relatively stable for a month, even when stored at 37°c, which is a warmer temperature than recommended by the manufacturer  <cit> .
with these advantages, lamp has the potential to be used even in clinical laboratories often poorly equipped, facing problems of constant electricity supply in tropical and sub-tropical countries where heartwater is endemic.
the purpose of the present study was to develop lamp assays for the detection of e. ruminantium and to evaluate the diagnostic sensitivity and specificity of these assays using a panel of bacterial dna samples, quantitated plasmid standards, and field samples derived from both animal blood and ticks.
the newly developed lamp assays successfully detected e. ruminantium with rapidity, specificity, and high sensitivity.
RESULTS
optimization of lamp
the reactions for both pcs <dig> and sodb lamp were performed under isothermal conditions at a range of  <dig> to 66°c using plasmid dna  for  <dig> min, with monitoring of the turbidity.
although amplifications with the lamp assays were observed at all temperatures tested, the reactions reached the threshold value  with the shortest incubation times at 61°c for pcs <dig> and 63°c for sodb .
no nonspecific amplification was detected for the negative cell culture until after at least  <dig> min incubation.
thus, subsequent lamp reactions were conducted at these temperatures for  <dig> min.
sensitivity of lamp assays
the sensitivities of pcs <dig> and sodb lamp assays are shown in figure 1a, and 1b, respectively.
a plot of the threshold time versus the log of the initial template copy number showed a linear regression, with statistically significant regression coefficients .
the detection limits for these assays, using a positive turbidity signal as the indicator, were  <dig> copies for pcs <dig> and  <dig> copies for sodb lamp.
alternative detection methods included agarose gel electrophoresis of the lamp products, which displayed the typical ladder-like pattern , as well as the detection of double stranded lamp products using gel-red .
with smaller amounts of dna in triplicate assays,  <dig> copies of pcs <dig> was amplified once, with a threshold time of  <dig>  min, and  <dig> copy of sodb was amplified twice with threshold times of  <dig>  and  <dig>  min.
specificity of lamp assays
the specificity of pcs <dig> and sodb lamp assays was evaluated by using the genomic dna of  <dig> known e. ruminantium isolates and five closely related species of anaplasmataceae: e. canis, e. chaffeensis, anaplasma centrale, a. marginale, and a. phagocytophilum.
all isolates of e. ruminantium were positive in both lamp assays, the pcs <dig> real-time pcr and the pcs <dig> pcr; whereas the pcs <dig> pcr was cross-reactive with both e. canis and e. chaffeensis .
inhibitory effect of dna preparation purified from bovine blood or ticks
in order to access inhibitory effects of components present in field samples, mixtures of standard plasmid dna and dna extracts from bovine blood and amblyomma variegatum were tested by the lamp assays.
when dna extracts from bovine blood were added to the templates, both pcs <dig> and sodb lamp could not detect  <dig> copies in two samples, which is in accordance with real-time pcr .
when dna extracts from a. variegatum were added to the templates, both pcs <dig> and sodb lamp failed in detecting  <dig> copies in all five samples, while real-time pcr could detect in four.
the pcs <dig> pcr using the kapa blood pcr kit detected more positives than the pcs <dig> pcr using the amplitaq gold pcr kit in the templates with  <dig> and  <dig> copies .
atotal no.
of samples positive by using the kapa blood pcr kit .
detection of e. ruminantium dna in field samples
a total of  <dig> a. variegatum ticks were collected in  <dig> sites in uganda and individually analyzed for the presence of e. ruminantium dna.
out of  <dig> ticks, including  <dig> males and  <dig> females,  <dig> ticks  were found positive with both pcs <dig> lamp and sodb lamp.
the pcs <dig> real-time pcr detected  <dig> positives, including the  <dig> lamp-positive ticks and an additional tick from dokolo, while pcs <dig> pcr could detect only  <dig> positives .
all the samples found positive with pcr were also positive with lamp.
the percent positive with lamp  was higher than with pcr  but slightly lower than with real-time pcr .
of the  <dig> bovine,  <dig> goat, and  <dig> lamb blood samples analyzed, two lamb samples were positive using pcr, real-time pcr, and lamp .
apcr was performed using kapa blood pcr kit.
bnd, not done.
cblood samples from sheep experimentally infected with e. ruminantium were used as positive controls.
dna, not applicable.
etotal no.
of ticks .
cross-reactivity of lamp with zoonotic ehrlichia in the usa
lamp assays were conducted with  <dig> amblyomma americanum dna samples from the usa that had previously tested positive for e. chaffeensis, e. ewingii, or pm ehrlichia .
both of the genetic clades of pm ehrlichia that have been described were represented among these samples.
all  <dig> samples tested negative using both lamp assays .
aehrlichia species were detected by previously described assays  <cit> .
bmap <dig> types; b <dig>  clade  <dig>  pme, and pmtn, represents the phylogenetic clade based on the sequence of major antigenic protein  <dig>  gene  <cit> .
cnr, not recorded.
dage was not recorded.
discussion
this report describes the development of two e. ruminantium-specific lamp assays based on the pcs <dig> and sodb genes.
the pcs <dig> region was the first target used for the genetic detection of e. ruminantium  <cit> .
subsequently, peter et al.
developed a pcr assay targeting pcs <dig> region with primers ab <dig> and ab <dig> for sensitive and specific detection of e. ruminantium  <cit> .
this assay was further evaluated for its reliability by the same authors  <cit>  and has been widely used by many researchers  <cit> .
because primers ab128/ <dig> could not amplify the divergent isolate of kümm <dig>  van heerden et al.
designed primers hh1f and hh2r in a highly conserved region of pcs <dig>  <cit> .
however, the major drawback of latter assay was cross-reactivity with closely related bacteria such as e. canis and e. chaffeensis, which were not detected by former assay  <cit> .
although pcs <dig> real-time pcr was also reported to be cross-reactive with e. canis and e. chaffeensis  <cit> , our study did not give the same results .
this inconsistency may be explained by the differences of sequence in pcs <dig> region between isolates as observed in e. ruminantium  <cit> .
thus, in this study, we have developed lamp assays based on not only pcs <dig> but also sodb because of its high degree of conservation among isolates.
the pairwise sequence identities calculated for pcs <dig> showed that the lowest pairwise identity for pcs <dig> sequences was  <dig> % , whereas that the lowest pairwise identity for the more conserved sodb gene was  <dig> %   <cit> .
this implies that sodb might be a more suitable target than pcs <dig> for the genetic detection of this species.
compared to the sequence of welgevonden isolate, the kümm <dig> differs by  <dig> out of  <dig> bp in the region targeted by the pcs <dig> lamp, while there is no sequence difference in the region targeted by sodb lamp .
although both pcs <dig> and sodb lamp detected all the e. ruminantium isolates tested in the present study, sodb lamp is more likely to detect previously unknown, divergent isolates of e. ruminantium.
thus, we concluded that sodb lamp is more suitable for detecting e. ruminantium and the diagnosis will be made more reliable in combination with pcs <dig> lamp.
the detection limits of the pcs <dig> and sodb lamp assays were  <dig> and  <dig> copies per reaction, respectively, which are at least 10-times more sensitive than that of conventional pcs <dig> pcr but slightly less sensitive than pcs <dig> real-time pcr  <cit> .
according to the instructions for lamp primer design software, the stability of primer end, especially 5' end of f1c/b1c and 3' end of f2/b <dig> as well as f3/b <dig>  is one of the crucial factors for designing proper lamp primers http://loopamp.eiken.co.jp/e/lamp/primer.html.
when lamp primers were designed for conserved pcs <dig> regions within isolates, only limited number of primer candidates were obtained initially .
therefore, we had to change the optimal values of parameters in the software for further designing pcs <dig> lamp primers.
in fact, an index for stability of primer, the dg value of the 5' end of the pcs <dig> b1c region , is above the value recommended by manufactures , which may explain lower sensitivities of pcs <dig> lamp than sodb lamp.
as is documented in several reports  <cit> , lamp showed relative tolerance to pcr inhibitors in blood, which was comparable to pcs <dig> real-time pcr .
however, lamp was clearly inhibited when dna extracts from a. variegatum were included in the reaction .
it is known that amblyomma tick tissue contains pcr-inhibitory elements which cannot be always removed during dna purification  <cit> .
thus, lamp is slightly less sensitive in the presence of such inhibitors in ticks compared to real-time pcr.
however, considering that real-time pcr is time-consuming and requires a thermal cycler with real-time monitoring and data analysis systems, which is expensive and can be relatively complicated to use, lamp has clear advantages over real-time pcr in terms of a practical system in a standard diagnostic laboratory, especially those in developing countries where the disease is prevalent.
in the present study, two sheep blood samples from a heartwater-endemic area tested positive by lamp .
domestic ruminants are known to occasionally harbor e. ruminantium without any clinical signs and to serve as reservoirs of the disease after recovery  <cit> .
previous reports demonstrated that pcr assays could detect the pathogen in the peripheral blood of clinically healthy animals in heartwater endemic areas  <cit> , indicating that a dna-based technique is useful even for the diagnosis of latent infection.
hence, lamp is a powerful tool not only for the epidemiological study of heartwater but also for the rapid and sensitive diagnosis of infected animals in the disease-endemic areas.
the simplest way of detecting lamp products is to inspect the white turbidity that results from magnesium pyrophosphate accumulation, as a by-product of the reaction, by naked eye  <cit> .
however, a small amount of this white precipitate is not always distinguishable from other white precipitates, such as proteins or carbohydrates, derived from the templates.
as an alternative method, this study employed a closed system, coupled with a double-stranded dna -binding dye, for low-cost detection of amplified dna .
the results obtained by this system were consistent with those obtained by gel electrophoresis .
since the detection can be accomplished in a closed system, without opening the reaction tubes, the risk of contamination is much lower than in gel electrophoresis or by adding dye at the end of the reaction.
theoretically, it should be possible to replace the gel-red tm dye we used with other dyes such as sybr green i  <cit> , ethidium bromide, evagreen  <cit> , and picogreen  <cit> , which are reported to be useful for the detection of lamp products.
as well documented by burridge et al., heartwater may easily be introduced into the american mainland through the introduction of infected ticks or animals from heartwater endemic areas  <cit> .
once imported, it is likely that the disease could become established because of the presence of local potential tick vectors  <cit> .
in order to prevent this pathogen from spreading into the usa, a screening test with high sensitivity and specificity is needed prior to the animal importation.
in this respect, the  <dig> dna samples from a. americanum harboring dna from ehrlichia species that are enzootic to the usa were found to be negative in lamp.
considering that the detection limits of the pcr assay used for the detection of ehrlichia species in a. americanum were  <dig> copies per reaction  <cit> , which is comparable to those of lamp assays, these samples were lamp-negative not because the dna concentrations were below the detection levels but probably because there were no cross reactions due to sequence mismatches or deletions in the targeted regions.
CONCLUSIONS
the lamp assays developed in this study allow rapid, sensitive, and specific detection of e. ruminantium.
although lamp reactions were inhibited in the presence of extracts from blood and ticks, the diagnostic sensitivity of lamp was higher than that of conventional pcr, when tested with field-collected ticks.
since lamp requires minimal time and equipment to perform, this technique can potentially be used in resource-poor settings where heartwater is endemic.
the lack of cross-reactivity with closely related ehrlichia species enhances its utility for active screening in areas under threat of the introduction of the disease.

 the rickettsial bacterium ehrlichia ruminantium is a causative agent of heartwater, the disease of ruminants transmitted by ticks of the genus amblyomma. heartwater is not only responsible for high economic losses in endemic countries, but is also suggested to be a potential emerging zoonosis since the pcr and sequence detection of the pathogen's presence in three fatal human cases although the cytological examination and bacterial isolation were not achieved. the disease is established in nearly all countries of sub-saharan africa and some islands of the caribbean, from where it threatens the american mainland. in the usa, three ehrlichia species, namely e. canis, e. chaffeensis, and e. ewingii, are known to exist. recently, panola mountain ehrlichia, which is closely related to e. ruminantium, was discovered as a novel zoonotic ehrlichia in the state of georgia. active surveillance using a reliable method which can discriminate e. ruminantium from these other ehrlichia species is an asset in preventing introduction of heartwater into the usa. in heartwater endemic countries, conventional diagnosis is based upon clinical signs and microscopic examination of post-mortem brain smears. as a more reliable and sensitive diagnostic method, several pcr-based assays have been developed for the detection of e. ruminantium, including conventional pcr, nested pcr, and real-time pcr. among them, the pcs real-time pcr taqman probe assay provides the best sensitivity with a detection limit of one gene copy per reaction, which is times higher than that of conventional pcs pcr. however, this assay was reported to cross-react with both e. chaffeensis and e. canis. moreover, although this assay performs well in the sensitive detection and quantification of e. ruminantium, it is not readily transferable to low-technology settings where there is limited access to expensive fluorescence detector based thermocyclers. loop-mediated isothermal amplification assay is a rapid dna amplification method originally developed by notomi et al., and it has been applied for the detection of viral, bacterial, fungal, and parasitic agents, but it has never previously been applied to rickettsial agents. the method requires a specially designed primer set that recognizes at least six independent regions of the target gene, which increases the specificity as well as the rapidity of the reaction. lamp results are visualized by turbidity that can be seen by the naked eye, and optionally by agarose gel electrophoresis or by addition of fluorescent dyes visualized under uv light. since the bst dna polymerase used in lamp allows strand displacement-dna synthesis, lamp reactions are performed under isothermal conditions using a simple incubator, such as a water bath or heating block. furthermore, lamp reagents are relatively stable for a month, even when stored at 37°c, which is a warmer temperature than recommended by the manufacturer. with these advantages, lamp has the potential to be used even in clinical laboratories often poorly equipped, facing problems of constant electricity supply in tropical and sub-tropical countries where heartwater is endemic. the purpose of the present study was to develop lamp assays for the detection of e. ruminantium and to evaluate the diagnostic sensitivity and specificity of these assays using a panel of bacterial dna samples, quantitated plasmid standards, and field samples derived from both animal blood and ticks. the newly developed lamp assays successfully detected e. ruminantium with rapidity, specificity, and high sensitivity.  optimization of lamp the reactions for both pcs and sodb lamp were performed under isothermal conditions at a range of to 66°c using plasmid dna for min, with monitoring of the turbidity. although amplifications with the lamp assays were observed at all temperatures tested, the reactions reached the threshold value with the shortest incubation times at 61°c for pcs and 63°c for sodb. no nonspecific amplification was detected for the negative cell culture until after at least min incubation. thus, subsequent lamp reactions were conducted at these temperatures for min. sensitivity of lamp assays the sensitivities of pcs and sodb lamp assays are shown in figure 1a, and 1b, respectively. a plot of the threshold time versus the log of the initial template copy number showed a linear regression, with statistically significant regression coefficients. the detection limits for these assays, using a positive turbidity signal as the indicator, were copies for pcs and copies for sodb lamp. alternative detection methods included agarose gel electrophoresis of the lamp products, which displayed the typical ladder-like pattern, as well as the detection of double stranded lamp products using gel-red. with smaller amounts of dna in triplicate assays, copies of pcs was amplified once, with a threshold time of min, and copy of sodb was amplified twice with threshold times of and min. specificity of lamp assays the specificity of pcs and sodb lamp assays was evaluated by using the genomic dna of known e. ruminantium isolates and five closely related species of anaplasmataceae: e. canis, e. chaffeensis, anaplasma centrale, a. marginale, and a. phagocytophilum. all isolates of e. ruminantium were positive in both lamp assays, the pcs real-time pcr and the pcs pcr; whereas the pcs pcr was cross-reactive with both e. canis and e. chaffeensis. inhibitory effect of dna preparation purified from bovine blood or ticks in order to access inhibitory effects of components present in field samples, mixtures of standard plasmid dna and dna extracts from bovine blood and amblyomma variegatum were tested by the lamp assays. when dna extracts from bovine blood were added to the templates, both pcs and sodb lamp could not detect copies in two samples, which is in accordance with real-time pcr. when dna extracts from a. variegatum were added to the templates, both pcs and sodb lamp failed in detecting copies in all five samples, while real-time pcr could detect in four. the pcs pcr using the kapa blood pcr kit detected more positives than the pcs pcr using the amplitaq gold pcr kit in the templates with and copies. atotal no. of samples positive by using the kapa blood pcr kit. detection of e. ruminantium dna in field samples a total of a. variegatum ticks were collected in sites in uganda and individually analyzed for the presence of e. ruminantium dna. out of ticks, including males and females, ticks were found positive with both pcs lamp and sodb lamp. the pcs real-time pcr detected positives, including the lamp-positive ticks and an additional tick from dokolo, while pcs pcr could detect only positives. all the samples found positive with pcr were also positive with lamp. the percent positive with lamp was higher than with pcr but slightly lower than with real-time pcr. of the bovine, goat, and lamb blood samples analyzed, two lamb samples were positive using pcr, real-time pcr, and lamp. apcr was performed using kapa blood pcr kit. bnd, not done. cblood samples from sheep experimentally infected with e. ruminantium were used as positive controls. dna, not applicable. etotal no. of ticks. cross-reactivity of lamp with zoonotic ehrlichia in the usa lamp assays were conducted with amblyomma americanum dna samples from the usa that had previously tested positive for e. chaffeensis, e. ewingii, or pm ehrlichia. both of the genetic clades of pm ehrlichia that have been described were represented among these samples. all samples tested negative using both lamp assays. aehrlichia species were detected by previously described assays. bmap types; b clade pme, and pmtn, represents the phylogenetic clade based on the sequence of major antigenic protein gene. cnr, not recorded. dage was not recorded. discussion this report describes the development of two e. ruminantium-specific lamp assays based on the pcs and sodb genes. the pcs region was the first target used for the genetic detection of e. ruminantium. subsequently, peter et al. developed a pcr assay targeting pcs region with primers ab and ab for sensitive and specific detection of e. ruminantium. this assay was further evaluated for its reliability by the same authors and has been widely used by many researchers. because primers ab128/ could not amplify the divergent isolate of kümm van heerden et al. designed primers hh1f and hh2r in a highly conserved region of pcs. however, the major drawback of latter assay was cross-reactivity with closely related bacteria such as e. canis and e. chaffeensis, which were not detected by former assay. although pcs real-time pcr was also reported to be cross-reactive with e. canis and e. chaffeensis, our study did not give the same results. this inconsistency may be explained by the differences of sequence in pcs region between isolates as observed in e. ruminantium. thus, in this study, we have developed lamp assays based on not only pcs but also sodb because of its high degree of conservation among isolates. the pairwise sequence identities calculated for pcs showed that the lowest pairwise identity for pcs sequences was %, whereas that the lowest pairwise identity for the more conserved sodb gene was %. this implies that sodb might be a more suitable target than pcs for the genetic detection of this species. compared to the sequence of welgevonden isolate, the kümm differs by out of bp in the region targeted by the pcs lamp, while there is no sequence difference in the region targeted by sodb lamp. although both pcs and sodb lamp detected all the e. ruminantium isolates tested in the present study, sodb lamp is more likely to detect previously unknown, divergent isolates of e. ruminantium. thus, we concluded that sodb lamp is more suitable for detecting e. ruminantium and the diagnosis will be made more reliable in combination with pcs lamp. the detection limits of the pcs and sodb lamp assays were and copies per reaction, respectively, which are at least 10-times more sensitive than that of conventional pcs pcr but slightly less sensitive than pcs real-time pcr. according to the instructions for lamp primer design software, the stability of primer end, especially 5' end of f1c/b1c and 3' end of f2/b as well as f3/b is one of the crucial factors for designing proper lamp primers http://loopamp.eiken.co.jp/e/lamp/primer.html. when lamp primers were designed for conserved pcs regions within isolates, only limited number of primer candidates were obtained initially. therefore, we had to change the optimal values of parameters in the software for further designing pcs lamp primers. in fact, an index for stability of primer, the dg value of the 5' end of the pcs b1c region, is above the value recommended by manufactures, which may explain lower sensitivities of pcs lamp than sodb lamp. as is documented in several reports, lamp showed relative tolerance to pcr inhibitors in blood, which was comparable to pcs real-time pcr. however, lamp was clearly inhibited when dna extracts from a. variegatum were included in the reaction. it is known that amblyomma tick tissue contains pcr-inhibitory elements which cannot be always removed during dna purification. thus, lamp is slightly less sensitive in the presence of such inhibitors in ticks compared to real-time pcr. however, considering that real-time pcr is time-consuming and requires a thermal cycler with real-time monitoring and data analysis systems, which is expensive and can be relatively complicated to use, lamp has clear advantages over real-time pcr in terms of a practical system in a standard diagnostic laboratory, especially those in developing countries where the disease is prevalent. in the present study, two sheep blood samples from a heartwater-endemic area tested positive by lamp. domestic ruminants are known to occasionally harbor e. ruminantium without any clinical signs and to serve as reservoirs of the disease after recovery. previous reports demonstrated that pcr assays could detect the pathogen in the peripheral blood of clinically healthy animals in heartwater endemic areas, indicating that a dna-based technique is useful even for the diagnosis of latent infection. hence, lamp is a powerful tool not only for the epidemiological study of heartwater but also for the rapid and sensitive diagnosis of infected animals in the disease-endemic areas. the simplest way of detecting lamp products is to inspect the white turbidity that results from magnesium pyrophosphate accumulation, as a by-product of the reaction, by naked eye. however, a small amount of this white precipitate is not always distinguishable from other white precipitates, such as proteins or carbohydrates, derived from the templates. as an alternative method, this study employed a closed system, coupled with a double-stranded dna -binding dye, for low-cost detection of amplified dna. the results obtained by this system were consistent with those obtained by gel electrophoresis. since the detection can be accomplished in a closed system, without opening the reaction tubes, the risk of contamination is much lower than in gel electrophoresis or by adding dye at the end of the reaction. theoretically, it should be possible to replace the gel-red tm dye we used with other dyes such as sybr green i, ethidium bromide, evagreen, and picogreen, which are reported to be useful for the detection of lamp products. as well documented by burridge et al., heartwater may easily be introduced into the american mainland through the introduction of infected ticks or animals from heartwater endemic areas. once imported, it is likely that the disease could become established because of the presence of local potential tick vectors. in order to prevent this pathogen from spreading into the usa, a screening test with high sensitivity and specificity is needed prior to the animal importation. in this respect, the dna samples from a. americanum harboring dna from ehrlichia species that are enzootic to the usa were found to be negative in lamp. considering that the detection limits of the pcr assay used for the detection of ehrlichia species in a. americanum were copies per reaction, which is comparable to those of lamp assays, these samples were lamp-negative not because the dna concentrations were below the detection levels but probably because there were no cross reactions due to sequence mismatches or deletions in the targeted regions.  the lamp assays developed in this study allow rapid, sensitive, and specific detection of e. ruminantium. although lamp reactions were inhibited in the presence of extracts from blood and ticks, the diagnostic sensitivity of lamp was higher than that of conventional pcr, when tested with field-collected ticks. since lamp requires minimal time and equipment to perform, this technique can potentially be used in resource-poor settings where heartwater is endemic. the lack of cross-reactivity with closely related ehrlichia species enhances its utility for active screening in areas under threat of the introduction of the disease. 
lamp detected more positive samples than conventional pcr but less than real-time pcr, when tested with field samples collected in sub-saharan countries. the detection limits for each assay were copies for pcs and copies for sodb, which is at least times higher than that of the conventional pcs pcr assay. due to its simplicity and specificity, lamp has the potential for use in resource-poor settings and also for active screening of e. ruminantium in both heartwater-endemic areas and regions that are at risk of contracting the disease. the disease is distributed in nearly all of sub-saharan africa and some islands of the caribbean, from where it threatens the american mainland. the rickettsial bacterium ehrlichia ruminantium is the causative agent of heartwater, a potential zoonotic disease of ruminants transmitted by ticks of the genus amblyomma. this report describes the development of two different loop-mediated isothermal amplification assays for sensitive and specific detection of e. ruminantium. two sets of lamp primers were designed from the pcs and sodb genes. the assays detected different isolates of e. ruminantium from geographically distinct countries as well as two attenuated vaccine isolates.
 the rickettsial bacterium ehrlichia ruminantium is the causative agent of heartwater, a potential zoonotic disease of ruminants transmitted by ticks of the genus amblyomma. the disease is distributed in nearly all of sub-saharan africa and some islands of the caribbean, from where it threatens the american mainland. this report describes the development of two different loop-mediated isothermal amplification assays for sensitive and specific detection of e. ruminantium.  two sets of lamp primers were designed from the pcs and sodb genes. the detection limits for each assay were copies for pcs and copies for sodb, which is at least times higher than that of the conventional pcs pcr assay. dna amplification was completed within min. the assays detected different isolates of e. ruminantium from geographically distinct countries as well as two attenuated vaccine isolates. no cross-reaction was observed with genetically related rickettsiales, including zoonotic ehrlichia species from the usa. lamp detected more positive samples than conventional pcr but less than real-time pcr, when tested with field samples collected in sub-saharan countries.  due to its simplicity and specificity, lamp has the potential for use in resource-poor settings and also for active screening of e. ruminantium in both heartwater-endemic areas and regions that are at risk of contracting the disease. 