BACKGROUND
hemotropic mycoplasmas  are uncultivated, small, pleomorphic, wall-less bacteria that parasitize on the surface of animal erythrocytes .
hemoplasmas have now been reclassified into the mycoplasma genus based on 16s rrna gene sequence.
these pathogens can cause mild to severe hemolytic anemia, icterus, ill-thrift, infertility, and poor weight gain, but death is rare in infected adults .
worldwide, hemoplasmas have been reported to affect livestock , companion animals , wildlife  <cit> , and humans .
mycoplasma ovis , which could elicit major health problems and high mortality in lambs, has been identified in sheep and goats .
in  <dig>  a novel hemoplasma, a 17 bp long deletion and an overall the 16s rna gene sequence identity of only 97% compared to m. ovis, has been detected in hungary.
subsequent studies identified this new hemoplasma as ‘candidatus m. haemovis’  <cit> .
subsequently, complete genome sequencing conducted by deshuillers et al.
revealed that m. ovis strain michigan contains two copies of 16s rrna gene, one corresponding to m. ovis, and the other to ‘candidatus m. haemovis’  <cit> .
therefore, deshuillers et al.
and other researchers can speculate that m. ovis and ‘candidatus m. haemovis’ may exist as a single species with two copies of 16s rrna gene or as two different species  <cit> .
with an increasing number of hemoplasmosis clinical cases, many countries have suffered extensive losses of livestock.
however, few epidemiological surveys on hemoplasmosis in sheep and goats have been conducted during the last decade.
the prevalence of m. ovis was  <dig> %  in small ruminants in north africa  <cit> , 20%  in goats in switzerland  <cit> , 87%  in captive cervids in brazil  <cit>  and  <dig> %  in free-living japanese serows  <cit> .
surprisingly, a 49-year-old veterinarian from texas of usa was found to be co-infected by two m. ovis variants, m. ovis and ‘candidatus m. haemovis’.
in china, m. ovis was found in  <dig> %  and  <dig> %  in small ruminants from hubei province and chongqing city, respectively  <cit> .
however, understanding of the molecular epidemiology of hemoplasmas  is limited in sheep and goats.
and ‘candidatus m. haemovis’ was poorly studied throughout the world and had never been detected in china until now.
thus, the aim of the present study was to determine the molecular prevalence of hemoplasmas, including m. ovis and ‘candidatus m. haemovis’ in sheep and goats from seven provinces and one autonomous region of china.
methods
specimen collection
a total of  <dig> edta-anticoagulated blood samples were collected from jugular vein of sheep and goats in henan, guizhou, shanxi, shaanxi, yunnan, qinghai, heilongjiang provinces and the inner mongolia autonomous region from march  <dig> to may  <dig> .fig.
<dig> collection sites  in china
for each animal, one blood sample was collected into an edta tube marked with breed, gender, age, and feeding habit of the animal.
the animals have no obvious clinical symptoms during blood sampling.
the specimens were then transported in an ice box to the laboratory for further processing and analysis.
dna extraction
for each specimen, dna was extracted from 500 μl of anticoagulated whole blood using a genome dna extraction kit  according to the manufacturer’s instructions.
dna was eluted from the column with 200 μl tris-edta buffer.
after dna extraction, dna concentration and quality was measured using absorbance ratio between 260/280 nm .
and all the specimens were subjected to a gapdh gene conventional pcr assay as described previously to confirm the presence of amplifiable dna and exclude pcr inhibition  <cit> .
dna was stored at −20 °c until pcr amplification.
nested pcr
all samples were tested at least three times for hemoplasmas  dna using a nested pcr assay that amplified an approximately 500 bp fragment of the 16s rrna gene  <cit> .
the base sequences of four primers  are shown in table  <dig> and the binding sites of four primers are shown in additional file 1: figure s <dig>  first-round pcr was performed in a total volume of 25 μl, containing  <dig>  μl of 10 × la pcr buffer ii ,  <dig>  μl of dntp mixture ,  <dig>  μl of each a <dig> and a <dig> primer ,  <dig>  u of la taq polymerase ,  <dig>  μl of double distilled water, and  <dig>  μl of dna template.
cycling conditions were: 94 °c for 5 min, followed by 30 cycles of 94 °c for 30 s, 61 °c for 30 s, and 72 °c for 1 min, with a final extension at 72 °c for 7 min.
second-round nested pcr contained  <dig>  μl of 10 × pcr buffer,  <dig>  μl of dntp mixture ,  <dig>  μl of each b <dig> and b <dig> primer ,  <dig>  u of r taq polymerase ,  <dig>  μl of double distilled water, and  <dig>  μl of the first-round pcr product .
cycling conditions were: 94 °c for 5 min, followed by 35 cycles of 94 °c for 30 s, 59 °c for 30 s, and 72 °c for 45 s, with a final extension at 72 °c for 7 min.
a positive control for m. ovis from a naturally infected boer goat preserved by our laboratory was used in all reactions, while double distilled water was used as a negative control.
secondary pcr products were examined by 1% agarose gel electrophoresis and visualized after gelred  staining.table  <dig> primers used for amplification of the 16s rrna gene of hemoplasmas
pcr for 16s rrna gene and sequencing
one hundred three positive specimens representative of different hosts, farms/flocks and geographic locations were selected and the nearly complete 16s rrna gene sequences were amplified using the protocol described by grazziotin et al.
<cit> .
primers are shown in table  <dig>  the 25 μl pcr mixture is the same as first-round pcr mixture of the nested pcr.
pcr products were purified using montage pcr filters  and sequenced using a bigdye terminator v <dig>  cycle sequencing kit  on an abi  <dig> dna analyzer .
the nucleotide sequences were confirmed by bidirectional sequencing and by sequencing a new pcr product if necessary.
phylogenetic analysis
nucleotide sequences of the nearly complete 16s rrna gene together with reference sequences downloaded from genbank were aligned using clustalx  <dig>   with further adjustments made manually as necessary.
and then mega  <dig>   software was applied to conduct phylogenetic and molecular evolutionary analysis.
a bootstrap phylogenetic tree demonstrating the relationship of m. ovis and ‘candidatus m. haemovis’ genotypes to other hemoplasma species was created by the neighbor-joining method using a distance matrix corrected for nucleotide substitutions based on the kimura 2-parameter model.
a bootstrap analysis was used to assess the robustness of the clusters using  <dig> replicates.
statistical analysis
positive rates were compared using the chi-square test.
differences were considered statistically significant at p <  <dig> .
analyses were performed using quickcalcs software .
the odds ratios  of the univariate analysis were calculated using measures of association along with 95% confidence intervals .
nucleotide sequence accession numbers
the four novel sequences determined in this study were deposited in genbank under accession numbers: ku <dig>  ku <dig>  ku <dig> and ku <dig>
RESULTS
molecular prevalence of hemoplasmas  in sheep and goats
as shown in fig.
<dig>  of the  <dig> blood specimens from sheep and goats,  <dig>  were found to be positive for hemoplasmas  by pcr amplification of the 16s rrna gene.
and the positive samples distributed in  <dig> studied farms from seven provinces and one aut.
reg.. in the  <dig> sheep and goats from heilongjiang province,  <dig>  were identified as hemoplasmas  -positive, while just two  of the  <dig> animals in the inner mongolia aut.
reg.
was positive.
on the other hand, the animals in three farms, including one in henan province and two from yunnan province were 100% positive for hemoplasmas  .
the infection rates of hemoplasmas  in sheep and goats in heilongjiang  and yunnan  were significantly higher than that of other provinces/aut.
reg.. a higher prevalence was observed in grazing sheep and goats  than household sheep and goats .
male animals had significantly higher hemoplasmas  infection rates, at  <dig> %, than females .
there were no significant differences between prevalence in different age groups of sheep and goats as well as that between sheep  and goats  .
the potential risk factors associated to percentage of infection with hemoplasmas were determined by statistical calculations.
among animal age, gender, species and feeding habits, gender  and the use of grazing  were identified as potential risk factors for hemoplasmas infection  .table  <dig> factors associated with positive hemoplasmas  infection in sheep and goats in china
p
sequence analysis of 16s rrna gene
sequencing of the nearly complete 16s rrna gene was successful for the  <dig> randomly selected positive specimens.
among them, analysis of the 16s rrna gene sequences identified m. ovis  and ‘candidatus m. haemovis’ .
the origin of the  <dig> positive specimens was shown in additional file 1: table s <dig>  after alignment, the obtained  <dig> 16s rrna gene sequences formed four sequence clusters ku <dig>  ku <dig>  ku <dig> and ku <dig>
ku <dig>  and ku <dig>  genotypes had  <dig> % and  <dig> % identity, respectively, to m. ovis from sheep in the usa , and  <dig> % and  <dig> % identity, respectively, to a human m. ovis isolate form usa .
both genotypes shared a high degree of identity  with another human m. ovis isolate , while, lower identity of them with sheep ‘candidatus m. haemovis’ genotypes from japan   were identified.
however, the other two genotypes ku <dig>  and ku <dig>  were closely related to ‘candidatus m. haemovis’ , with  <dig> % and  <dig> % identities, respectively, yet, their identities with m. ovis  were  <dig> % and  <dig> %, respectively.
based on the 16s rrna gene sequence analysis, the four novel genotypes in this study differed from m. ovis  at several nucleotide positions along the full-length of the gene .table  <dig> 16s rrna gene alignment from two m. ovis genotypes  and two ‘candidatus m. haemovis’ genotypes  and their comparison with two hemotropic mycoplasma sequences
positiona
aposition numbers given with respect to m. ovis
*identical to the reference sequence; −, base deletion
phylogenetic analysis
phylogenetic analysis revealed that the four novel genotypes accompanied by five other ‘candidatus m. haemovis’ , six other m. ovis  as well as one mycoplasma spp.
formed a separate clade.
in addition, ‘candidatus m. haemovis’ and m. ovis genotypes fell into two different groups.
the two genotypes ku <dig> and ku <dig>  representing 56 m. ovis sequences from the present study, were grouped in the same clade with two human genotypes from usa  .fig.
<dig> phylogenetic relationships of mycoplasma ovis and ‘candidatus m. haemovis’ genotypes identified here and other hemotropic mycoplasmas.
phylogeny was inferred with a neighbor-joining analysis of the nearly complete 16s rrna gene sequences based on distances calculated with the kimura 2-parameter model.
mycoplasma pneumoniae was used as an outgroup.
bootstrap values >50% from  <dig> replicates are shown on the nodes.
the genotypes detected in this study are shown as triangles
discussion
globally, hemotropic mycoplasma spp.
are emerging or re-emerging zoonotic pathogens that affect numerous animal species including humans, potentially causing loss of livestock and public health concern.
m. ovis is prevalent worldwide, and is causative of hemolytic anemia in sheep and goats  <cit> .
in the present study, the prevalence of hemoplasmas  in sheep and goats was found to be  <dig> % .
a similar prevalence of m. ovis was previously reported in goats in hubei, china   <cit> , while a lower rate was detected in sheep in tunisia   <cit> .
a higher m. ovis prevalence was previously found in sheep in malaysia   <cit> , argentina   <cit> , and hungary   <cit> .
the differences in prevalence between this study and others  <cit>  may be caused by discrepancy in the age groups studied, as well as the number of animals, detection methods, and geographic and ecological environments.
in this study, there is a striking difference in prevalence between the north-central sampling sites  versus the southern and eastern sites .
the climate type of north-central sampling sites is monsoon climate of medium latitudes, whereas southern sites are subtropical monsoon climate.
invertebrate vector abundance and vegetation species in north-central sampling sites is relatively lower than southern sites.
therefore, we speculated the different climates, vegetation species and invertebrate vector abundance factors that may contribute to the apparent difference in prevalence in different provinces.
it was reported that the m. ovis prevalence was significantly higher in young sheep than adults  by rjeibi et al.
<cit> .
however, in the present study, there was no significant difference in hemoplasmas  infection rates between different age groups of sheep and goats.
nevertheless, significant differences were detected among the prevalence of hemoplasmas  according to animal gender and feeding habits of the host animals, although no differences were observed between samples obtained from different genders by song et al.
<cit> .
in this study, potential risk factors were identified as animal gender and feeding habits of the host animals, providing useful information for control of the hemoplasmosis.
grazing flocks are more likely to be exposed to blood-sucking arthropods than household flocks.
household flocks received good feeding and management, which were thought to be important for the prevention from various diseases.
therefore, infections of hemoplasmas may be transmitted by blood-sucking arthropods and reusing needles during herd immunization in china.
‘candidatus m. haemovis’ was first demonstrated in a sheep flock with fatal hemolytic anemia in hungary  <cit> , following studies reported the presence of both m. ovis and ‘candidatus m. haemovis’ dnas in sheep in hungary  <cit> , japan  <cit> , and the usa  <cit> , in goats in switzerland  <cit> , and in capricornis crispus in japan  <cit> .
however, there was no report documented the existence of m. ovis and ‘candidatus m. haemovis’ in sheep and goats in china.
therefore, detection of hemoplasmas  by nested pcr and sequence analyses in the present study represents the first molecular evidence of the occurrence of ‘candidatus m. haemovis’ in sheep and goats in china.
phylogenetic analysis revealed that ‘candidatus m. haemovis’ and m. ovis belong to the same clade.
16s rrna gene homology between m. ovis and ‘candidatus m. haemovis’ in this study was very high, at  <dig> – <dig> % , revealing the close genetic relationship between them, which suggested that ‘candidatus m. haemovis’ might not be a new hemoplasma species, but another copy of m. ovis 16s rrna gene, which was validated by deshuillers et al.’s report that m. ovis strain michigan has two different copies of the 16s rrna gene.
the detection of multiple different 16s rrna gene sequences in a veterinarian in texas  <cit> , in a severe anemia sheep in japan  <cit> , in goats in switzerland  <cit>  and in sheep in hungary  <cit> , might also be explained by the existence of multiple copies of the 16s rrna gene within a single strain of m. ovis, each having a variable genetic composition, as reported for other bacteria  <cit> .
in previous studies, only five hemotropic mycoplasma spp., m. haemofelis-like  <cit> , m. suis-like  <cit> , m. ovis  <cit> , ‘candidatus mycoplasma haemohominis’  <cit> , and ‘candidatus mycoplasma haematoparvum’  <cit>  organisms, were described to be infected by human beings.
sykes et al.
reported the first case of human infection with hemotropic mycoplasma  in a veterinarian in texas.
additionally, among the  <dig> hemotropic mycoplasma spp.
identified from  <dig> patients, m. ovis-like organism was the most prevalent  <cit> , a species which was mainly found in small ruminants, such as deer  <cit> , goats  <cit> , sheep  <cit> , and japanese serows  <cit> .
it is noteworthy that the nucleotide sequences of m. ovis  in the present study had highest homology with m. ovis  from humans .
the high homology of the m. ovis genotypes obtained in the present study with human genotypes observed here may provide additional evidence for the zoonotic potential of m. ovis, which were raised by sykes et al.
and then verified by maggi et al.
CONCLUSIONS
this study provides further evidence about the prevalence of hemoplasmas , and presents the first detection of ‘candidatus m. haemovis’ in sheep and goats in china.
our data showed a high prevalence and widespread distribution of hemoplasmas  in sheep and goats.
further studies are needed to evaluate the pathogenicity of ‘candidatus m. haemovis’ in sheep and goats, as well as the zoonotic potential of m. ovis.
additional file
additional file 1: table s <dig>  prevalence of hemoplasmas  of sheep and goats in different farms.
table s <dig>  the origins of the  <dig> selected positive specimens.
figure s <dig>  collection sites  in henan province.
figure s <dig>  the binding sites of nested pcr primers.
abbreviations
‘candidatus m. haemovis’‘candidatus mycoplasma haemovis’
16s rrna16s ribosomal rna
aut.
reg.autonomous region
b. henselae
bartonella henselae
dnadeoxyribonucleic acid
edtaethylenediaminetetraacetic acid
hivhuman immunodeficiency virus
m. haemofelis
mycoplasma haemofelis
m. ovis
mycoplasma ovis
m. suis
mycoplasma suis
pcrpolymerase chain reaction
electronic supplementary material
the online version of this article  contains supplementary material, which is available to authorized users.

 hemotropic mycoplasmas are uncultivated, small, pleomorphic, wall-less bacteria that parasitize on the surface of animal erythrocytes. hemoplasmas have now been reclassified into the mycoplasma genus based on 16s rrna gene sequence. these pathogens can cause mild to severe hemolytic anemia, icterus, ill-thrift, infertility, and poor weight gain, but death is rare in infected adults. worldwide, hemoplasmas have been reported to affect livestock, companion animals, wildlife, and humans. mycoplasma ovis, which could elicit major health problems and high mortality in lambs, has been identified in sheep and goats. in a novel hemoplasma, a 17 bp long deletion and an overall the 16s rna gene sequence identity of only 97% compared to m. ovis, has been detected in hungary. subsequent studies identified this new hemoplasma as ‘candidatus m. haemovis’. subsequently, complete genome sequencing conducted by deshuillers et al. revealed that m. ovis strain michigan contains two copies of 16s rrna gene, one corresponding to m. ovis, and the other to ‘candidatus m. haemovis’. therefore, deshuillers et al. and other researchers can speculate that m. ovis and ‘candidatus m. haemovis’ may exist as a single species with two copies of 16s rrna gene or as two different species. with an increasing number of hemoplasmosis clinical cases, many countries have suffered extensive losses of livestock. however, few epidemiological surveys on hemoplasmosis in sheep and goats have been conducted during the last decade. the prevalence of m. ovis was % in small ruminants in north africa, 20% in goats in switzerland, 87% in captive cervids in brazil and % in free-living japanese serows. surprisingly, a 49-year-old veterinarian from texas of usa was found to be co-infected by two m. ovis variants, m. ovis and ‘candidatus m. haemovis’. in china, m. ovis was found in % and % in small ruminants from hubei province and chongqing city, respectively. however, understanding of the molecular epidemiology of hemoplasmas is limited in sheep and goats. and ‘candidatus m. haemovis’ was poorly studied throughout the world and had never been detected in china until now. thus, the aim of the present study was to determine the molecular prevalence of hemoplasmas, including m. ovis and ‘candidatus m. haemovis’ in sheep and goats from seven provinces and one autonomous region of china. methods specimen collection a total of edta-anticoagulated blood samples were collected from jugular vein of sheep and goats in henan, guizhou, shanxi, shaanxi, yunnan, qinghai, heilongjiang provinces and the inner mongolia autonomous region from march to may.fig. collection sites in china for each animal, one blood sample was collected into an edta tube marked with breed, gender, age, and feeding habit of the animal. the animals have no obvious clinical symptoms during blood sampling. the specimens were then transported in an ice box to the laboratory for further processing and analysis. dna extraction for each specimen, dna was extracted from 500 μl of anticoagulated whole blood using a genome dna extraction kit according to the manufacturer’s instructions. dna was eluted from the column with 200 μl tris-edta buffer. after dna extraction, dna concentration and quality was measured using absorbance ratio between 260/280 nm. and all the specimens were subjected to a gapdh gene conventional pcr assay as described previously to confirm the presence of amplifiable dna and exclude pcr inhibition. dna was stored at −20 °c until pcr amplification. nested pcr all samples were tested at least three times for hemoplasmas dna using a nested pcr assay that amplified an approximately 500 bp fragment of the 16s rrna gene. the base sequences of four primers are shown in table and the binding sites of four primers are shown in additional file 1: figure s first-round pcr was performed in a total volume of 25 μl, containing  μl of 10 × la pcr buffer ii,  μl of dntp mixture,  μl of each a and a primer,  u of la taq polymerase,  μl of double distilled water, and  μl of dna template. cycling conditions were: 94 °c for 5 min, followed by 30 cycles of 94 °c for 30 s, 61 °c for 30 s, and 72 °c for 1 min, with a final extension at 72 °c for 7 min. second-round nested pcr contained  μl of 10 × pcr buffer,  μl of dntp mixture,  μl of each b and b primer,  u of r taq polymerase,  μl of double distilled water, and  μl of the first-round pcr product. cycling conditions were: 94 °c for 5 min, followed by 35 cycles of 94 °c for 30 s, 59 °c for 30 s, and 72 °c for 45 s, with a final extension at 72 °c for 7 min. a positive control for m. ovis from a naturally infected boer goat preserved by our laboratory was used in all reactions, while double distilled water was used as a negative control. secondary pcr products were examined by 1% agarose gel electrophoresis and visualized after gelred staining.table primers used for amplification of the 16s rrna gene of hemoplasmas pcr for 16s rrna gene and sequencing one hundred three positive specimens representative of different hosts, farms/flocks and geographic locations were selected and the nearly complete 16s rrna gene sequences were amplified using the protocol described by grazziotin et al.. primers are shown in table the 25 μl pcr mixture is the same as first-round pcr mixture of the nested pcr. pcr products were purified using montage pcr filters and sequenced using a bigdye terminator v  cycle sequencing kit on an abi dna analyzer. the nucleotide sequences were confirmed by bidirectional sequencing and by sequencing a new pcr product if necessary. phylogenetic analysis nucleotide sequences of the nearly complete 16s rrna gene together with reference sequences downloaded from genbank were aligned using clustalx with further adjustments made manually as necessary. and then mega software was applied to conduct phylogenetic and molecular evolutionary analysis. a bootstrap phylogenetic tree demonstrating the relationship of m. ovis and ‘candidatus m. haemovis’ genotypes to other hemoplasma species was created by the neighbor-joining method using a distance matrix corrected for nucleotide substitutions based on the kimura 2-parameter model. a bootstrap analysis was used to assess the robustness of the clusters using replicates. statistical analysis positive rates were compared using the chi-square test. differences were considered statistically significant at p < . analyses were performed using quickcalcs software. the odds ratios of the univariate analysis were calculated using measures of association along with 95% confidence intervals. nucleotide sequence accession numbers the four novel sequences determined in this study were deposited in genbank under accession numbers: ku ku ku and ku  molecular prevalence of hemoplasmas in sheep and goats as shown in fig. of the blood specimens from sheep and goats, were found to be positive for hemoplasmas by pcr amplification of the 16s rrna gene. and the positive samples distributed in studied farms from seven provinces and one aut. reg.. in the sheep and goats from heilongjiang province, were identified as hemoplasmas -positive, while just two of the animals in the inner mongolia aut. reg. was positive. on the other hand, the animals in three farms, including one in henan province and two from yunnan province were 100% positive for hemoplasmas. the infection rates of hemoplasmas in sheep and goats in heilongjiang and yunnan were significantly higher than that of other provinces/aut. reg.. a higher prevalence was observed in grazing sheep and goats than household sheep and goats. male animals had significantly higher hemoplasmas infection rates, at %, than females. there were no significant differences between prevalence in different age groups of sheep and goats as well as that between sheep and goats. the potential risk factors associated to percentage of infection with hemoplasmas were determined by statistical calculations. among animal age, gender, species and feeding habits, gender and the use of grazing were identified as potential risk factors for hemoplasmas infection.table factors associated with positive hemoplasmas infection in sheep and goats in china p sequence analysis of 16s rrna gene sequencing of the nearly complete 16s rrna gene was successful for the randomly selected positive specimens. among them, analysis of the 16s rrna gene sequences identified m. ovis and ‘candidatus m. haemovis’. the origin of the positive specimens was shown in additional file 1: table s after alignment, the obtained 16s rrna gene sequences formed four sequence clusters ku ku ku and ku ku and ku genotypes had % and % identity, respectively, to m. ovis from sheep in the usa, and % and % identity, respectively, to a human m. ovis isolate form usa. both genotypes shared a high degree of identity with another human m. ovis isolate, while, lower identity of them with sheep ‘candidatus m. haemovis’ genotypes from japan were identified. however, the other two genotypes ku and ku were closely related to ‘candidatus m. haemovis’, with % and % identities, respectively, yet, their identities with m. ovis were % and %, respectively. based on the 16s rrna gene sequence analysis, the four novel genotypes in this study differed from m. ovis at several nucleotide positions along the full-length of the gene.table 16s rrna gene alignment from two m. ovis genotypes and two ‘candidatus m. haemovis’ genotypes and their comparison with two hemotropic mycoplasma sequences positiona aposition numbers given with respect to m. ovis *identical to the reference sequence; −, base deletion phylogenetic analysis phylogenetic analysis revealed that the four novel genotypes accompanied by five other ‘candidatus m. haemovis’, six other m. ovis as well as one mycoplasma spp. formed a separate clade. in addition, ‘candidatus m. haemovis’ and m. ovis genotypes fell into two different groups. the two genotypes ku and ku representing 56 m. ovis sequences from the present study, were grouped in the same clade with two human genotypes from usa.fig. phylogenetic relationships of mycoplasma ovis and ‘candidatus m. haemovis’ genotypes identified here and other hemotropic mycoplasmas. phylogeny was inferred with a neighbor-joining analysis of the nearly complete 16s rrna gene sequences based on distances calculated with the kimura 2-parameter model. mycoplasma pneumoniae was used as an outgroup. bootstrap values >50% from replicates are shown on the nodes. the genotypes detected in this study are shown as triangles discussion globally, hemotropic mycoplasma spp. are emerging or re-emerging zoonotic pathogens that affect numerous animal species including humans, potentially causing loss of livestock and public health concern. m. ovis is prevalent worldwide, and is causative of hemolytic anemia in sheep and goats. in the present study, the prevalence of hemoplasmas in sheep and goats was found to be %. a similar prevalence of m. ovis was previously reported in goats in hubei, china, while a lower rate was detected in sheep in tunisia. a higher m. ovis prevalence was previously found in sheep in malaysia, argentina, and hungary. the differences in prevalence between this study and others may be caused by discrepancy in the age groups studied, as well as the number of animals, detection methods, and geographic and ecological environments. in this study, there is a striking difference in prevalence between the north-central sampling sites versus the southern and eastern sites. the climate type of north-central sampling sites is monsoon climate of medium latitudes, whereas southern sites are subtropical monsoon climate. invertebrate vector abundance and vegetation species in north-central sampling sites is relatively lower than southern sites. therefore, we speculated the different climates, vegetation species and invertebrate vector abundance factors that may contribute to the apparent difference in prevalence in different provinces. it was reported that the m. ovis prevalence was significantly higher in young sheep than adults by rjeibi et al.. however, in the present study, there was no significant difference in hemoplasmas infection rates between different age groups of sheep and goats. nevertheless, significant differences were detected among the prevalence of hemoplasmas according to animal gender and feeding habits of the host animals, although no differences were observed between samples obtained from different genders by song et al.. in this study, potential risk factors were identified as animal gender and feeding habits of the host animals, providing useful information for control of the hemoplasmosis. grazing flocks are more likely to be exposed to blood-sucking arthropods than household flocks. household flocks received good feeding and management, which were thought to be important for the prevention from various diseases. therefore, infections of hemoplasmas may be transmitted by blood-sucking arthropods and reusing needles during herd immunization in china. ‘candidatus m. haemovis’ was first demonstrated in a sheep flock with fatal hemolytic anemia in hungary, following studies reported the presence of both m. ovis and ‘candidatus m. haemovis’ dnas in sheep in hungary, japan, and the usa, in goats in switzerland, and in capricornis crispus in japan. however, there was no report documented the existence of m. ovis and ‘candidatus m. haemovis’ in sheep and goats in china. therefore, detection of hemoplasmas by nested pcr and sequence analyses in the present study represents the first molecular evidence of the occurrence of ‘candidatus m. haemovis’ in sheep and goats in china. phylogenetic analysis revealed that ‘candidatus m. haemovis’ and m. ovis belong to the same clade. 16s rrna gene homology between m. ovis and ‘candidatus m. haemovis’ in this study was very high, at – %, revealing the close genetic relationship between them, which suggested that ‘candidatus m. haemovis’ might not be a new hemoplasma species, but another copy of m. ovis 16s rrna gene, which was validated by deshuillers et al.’s report that m. ovis strain michigan has two different copies of the 16s rrna gene. the detection of multiple different 16s rrna gene sequences in a veterinarian in texas, in a severe anemia sheep in japan, in goats in switzerland and in sheep in hungary, might also be explained by the existence of multiple copies of the 16s rrna gene within a single strain of m. ovis, each having a variable genetic composition, as reported for other bacteria. in previous studies, only five hemotropic mycoplasma spp., m. haemofelis-like, m. suis-like, m. ovis, ‘candidatus mycoplasma haemohominis’, and ‘candidatus mycoplasma haematoparvum’ organisms, were described to be infected by human beings. sykes et al. reported the first case of human infection with hemotropic mycoplasma in a veterinarian in texas. additionally, among the hemotropic mycoplasma spp. identified from patients, m. ovis-like organism was the most prevalent, a species which was mainly found in small ruminants, such as deer, goats, sheep, and japanese serows. it is noteworthy that the nucleotide sequences of m. ovis in the present study had highest homology with m. ovis from humans. the high homology of the m. ovis genotypes obtained in the present study with human genotypes observed here may provide additional evidence for the zoonotic potential of m. ovis, which were raised by sykes et al. and then verified by maggi et al.  this study provides further evidence about the prevalence of hemoplasmas, and presents the first detection of ‘candidatus m. haemovis’ in sheep and goats in china. our data showed a high prevalence and widespread distribution of hemoplasmas in sheep and goats. further studies are needed to evaluate the pathogenicity of ‘candidatus m. haemovis’ in sheep and goats, as well as the zoonotic potential of m. ovis. additional file additional file 1: table s prevalence of hemoplasmas of sheep and goats in different farms. table s the origins of the selected positive specimens. figure s collection sites in henan province. figure s the binding sites of nested pcr primers. abbreviations ‘candidatus m. haemovis’‘candidatus mycoplasma haemovis’ 16s rrna16s ribosomal rna aut. reg.autonomous region b. henselae bartonella henselae dnadeoxyribonucleic acid edtaethylenediaminetetraacetic acid hivhuman immunodeficiency virus m. haemofelis mycoplasma haemofelis m. ovis mycoplasma ovis m. suis mycoplasma suis pcrpolymerase chain reaction electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. 
all blood samples were tested for hemoplasmas by nested pcr amplification based on 16s rrna gene. overall, specimens were shown to be hemoplasmas -positive by nested pcr amplification based on 16s rrna gene. a total of blood samples were collected from sheep and goats in seven provinces and one autonomous region of china. hemotropic mycoplasmas are emerging zoonotic pathogens with a worldwide distribution that can cause mild to severe hemolytic anemia, icterus, ill-thrift, infertility, and poor weight gain. the online version of this article contains supplementary material, which is available to authorized users. two of the four novel genotypes obtained in this study were closely related to m. ovis, while the other two genotypes had high identity with ‘candidatus m. haemovis’. thus, the aim of the present study was to determine the molecular prevalence of hemoplasmas, including m. ovis and ‘candidatus m. haemovis’ in sheep and goats from seven provinces and one autonomous region of china. positive specimens underwent nucleotide sequencing and phylogenetic analysis. sequencing of the nearly complete 16s rrna gene was successful for the randomly selected positive specimens from different farms in different sampling sites of china. among them, analysis of the 16s rrna gene sequences identified m. ovis and ‘candidatus m. haemovis’. however, understanding of the molecular epidemiology of hemoplasmas is limited in sheep and goats, and the hemoplasma strain/species/variant ‘candidatus m. haemovis’ was poorly studied throughout the world and had never been detected in china until now. remarkably, the genotype of m. ovis in sheep and goats in this study fell in a clade with two human hemoplasmas from usa and shared %– % with them. the prevalence in goats was %, and % in sheep, while that in grazing small ruminants was % and % in house feeding small ruminants. hemoplasmasmycoplasma ovis‘candidatus mycoplasma haemovis’small ruminants16s rrna genethe earmarked fund for china modern agro-industry technology research system nycytx-39ning changshen issue-copyright-statement© the author 2017
 hemotropic mycoplasmas are emerging zoonotic pathogens with a worldwide distribution that can cause mild to severe hemolytic anemia, icterus, ill-thrift, infertility, and poor weight gain. however, understanding of the molecular epidemiology of hemoplasmas is limited in sheep and goats, and the hemoplasma strain/species/variant ‘candidatus m. haemovis’ was poorly studied throughout the world and had never been detected in china until now. thus, the aim of the present study was to determine the molecular prevalence of hemoplasmas, including m. ovis and ‘candidatus m. haemovis’ in sheep and goats from seven provinces and one autonomous region of china. methods a total of blood samples were collected from sheep and goats in seven provinces and one autonomous region of china. all blood samples were tested for hemoplasmas by nested pcr amplification based on 16s rrna gene. positive specimens underwent nucleotide sequencing and phylogenetic analysis.  overall, specimens were shown to be hemoplasmas -positive by nested pcr amplification based on 16s rrna gene. the prevalence in goats was %, and % in sheep, while that in grazing small ruminants was % and % in house feeding small ruminants. sequencing of the nearly complete 16s rrna gene was successful for the randomly selected positive specimens from different farms in different sampling sites of china. among them, analysis of the 16s rrna gene sequences identified m. ovis and ‘candidatus m. haemovis’. two of the four novel genotypes obtained in this study were closely related to m. ovis, while the other two genotypes had high identity with ‘candidatus m. haemovis’. remarkably, the genotype of m. ovis in sheep and goats in this study fell in a clade with two human hemoplasmas from usa and shared %– % with them.  in this study, ‘candidatus m. haemovis’ was first detected in chinese sheep and goats and hemoplasmas are highly prevalent, and widely distributed in china. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords hemoplasmasmycoplasma ovis‘candidatus mycoplasma haemovis’small ruminants16s rrna genethe earmarked fund for china modern agro-industry technology research system nycytx-39ning changshen issue-copyright-statement© the author 2017 