BACKGROUND
toll-like receptors  have emerged as a major component of the vertebrate pattern recognition receptor  repertoire, and can recognize conserved pathogen-associated molecular patterns  of bacteria, fungi, viruses and parasites as well as some self-ligands, such as host dna in immune complexes and the malarial pigment hemozoin, , but not commensal microbes  <cit> .
upon activation by an agonist, tlrs induce the expression of a wide range of immunoregulatory and effector molecules  <cit> , which take part in the regulation of immune cell maturation and the clearance of pathogenic microorganisms  <cit> .
in humans and mice,four tlrs  are responsible for recognition of antigens from salmonella enteritidis ,a gram-negative bacteria, as well as via tlr <dig>  tlr <dig> and tlr2- <dig> in chicken  <cit> .furthermore, tlr <dig> dimerizes with tlr <dig> or tlr <dig>  enabling it to recognize microbial triacyl lipoproteins or diacyl lipopeptides found in mycoplasma, lipoteichoic acid of gram-positive bacteria or zymosan of yeasts  <cit> .however, caution is required in the interpretation of these findings as tlr <dig> may not recognize some agonists at physiologically relevant concentrations  <cit> .
recently, human tlr <dig> has also been shown to dimerize with tlr <dig> enabling activation by triacyl lipoproteins  <cit> .
chicken chtlr1la and b, and chtlr2a and b have been cloned and expressed in mammalian cells by several groups and were demonstrated to be surface- exposed  <cit> .
it was thought that some diseases, such as newcastle disease and h5n <dig> avian influenza, occurred only in domestic avian species, did not appear or had only very low morbidity in waterfowl  <cit> .
however, since  <dig>  serotype i avian paramyxoviruses  have caused high morbidity and mortality in geese during outbreaks in many provinces of china.
interestingly, recent studies have not determined any obvious variation in the gene sequences of apmv- <dig>  <cit> .
tlr <dig> genes has been identified in mammalian and nonmammalian vertebrates including chicken  <cit> .
however, it is not known whether geese also carry a homologue of mammalian tlr <dig> for recognizing the pathogens.
in the present study, we cloned and sequenced the goose tlr2- <dig>  analyzed its expression in several tissues and verified its lipopeptide-mediated activation in hek-293 t cells.
meanwhile, the expression of tlr2- <dig> in goose tissues after they were challenged with mycoplasma gallisepticum  or salmonella enteritidis  were also studied.
RESULTS
cloning of goose tlr2-1
r fragments of various sizes were subcloned into the vector pmd18-t, and their sequences were determined.
one of the clones was found to contain a sequence similar to the tir domain of chicken tlr2s.
a fragment of  <dig>  bp was obtained by 3′-race-pcr.
a 930-bp fragment was then amplified by 5′-race-pcr.
a  <dig>  bp nucleotide sequence representing the full-length, complete cdna of the goose tlr2- <dig> gene was obtained from overlapping cdna fragments.
the full-length cdna harbored an orf of  <dig>  bp, encoding a protein of  <dig> amino acids in length .
we aligned the determined nucleotide sequence from geese with sequences in genebank by blast search tool of national center for biotechnology information , and named the present gene as gtlr2- <dig>  the sequence was submitted to genbank and assigned the accession number genbank: jn <dig>  the untranslated regions  were 359 bp and 378 bp for the 5′-utr and 3′-utr, respectively.figure  <dig> the results of goose tlr2- <dig> gene cloning and eukaryotic expression.
electro-phoresis of rt-pcr and race-pcr products of the goose tlr2- <dig> gene.
lane  <dig> represents the 5′-race-pcr product, a 930-bp fragment.
lane  <dig> represents the 3′-race-pcr product, a  <dig>  bp fragment.
lane  <dig> represents the homologous cdna fragments from chicken tlr2- <dig>  forming a band of 2150 bp.
electrophoresis of the full-length tlr2-1encoding an open read frame, a fragment of  <dig>  bp.
immunoblotting of gtlr2- <dig> expressed in hek <dig> cells, showing a 103-kda band.
prediction of protein domains revealed that the putative amino acid sequence consisted of a signal peptide sequence encompassing the first  <dig> amino acid residues of the n-terminal region, nine leucine-rich repeat  domains, a leucine-rich repeat c-terminal  domain, a transmembrane domain and a 144-amino-acid toll-interleukin-receptor  domain at positions 649– <dig> of the carboxy-terminus .
highly conserved three subsections of the tir domain  is shown in figure  <dig> figure  <dig> nucleotide and deduced amino acid sequence of goose tlr2- <dig>  complete sequence of the full-length gtlr2- <dig> was deposited in the cdna library .
translated amino acid sequence is shown under the nucleotide sequence.
numbers to the left of each row refer to nucleotides.
the cleavage site for the putative signal peptide is indicated by an arrow.
leucine-rich repeat  domains are shaded in grey.
potential n-glycosylation sites are shown as square boxes.
the initiation codon  is underlined.
the predicted transmembrane segment is double underlined.
the translational stop site is indicated by an asterisk.
the cysteines critical for the maintenance of the structure of leucine-rich repeat c-terminal  are marked with coarse underlines.
lrr-typ motif is shown by wave lines.
tir motif is shown by a long box.
tissue distribution analyzed by quantitative rt-pcr
the expression of gtlr2- <dig> in geese organ tissues was analyzed by quantitative rt-pcr.
the level of gtlr2- <dig> mrna was highest in kidney, and moderate expression levels were seen in bursa, intestine, brain, stomach and heart.
the expression levels in the spleen and lung were relatively low .figure  <dig> relative gtlr2- <dig> expression levels in goose tissues.
the expression of the gtlr2- <dig> gene in  <dig> different tissues was analyzed by quantitative rt-pcr.
the data are presented as mean +/− sd, and were normalized using the β-actin gene and calculated by the delta ct method.
we compared the gtlr2- <dig> expression in indicated tissues to that in spleen .
“*” and “**”indicates that p ≤  <dig>  and p ≤  <dig>  for each test respectively.
malp- <dig> stimulation of hek- <dig> cells expressing goose tlr2- <dig> results in nf-κb activation
goose tlr2- <dig> was cloned downstream from a pcmv promoter with a his-tag fused at the carboxyl terminus.
expression of gtlr2- <dig> was confirmed by western blot analysis of hek- <dig> cells transiently transfected with the gtlr2- <dig> expression plasmid, showing a protein band with an expected size of  <dig> kd .
as the activation of tlrs results in its interaction with adaptor molecules and downstream signaling, leading to upregulation of genes with nf-κb elements, a nf-κb luciferase reporter system was used to determine whether the expressed recombinant goose tlr could be activated by exogenous macrophage-activating lipopeptide  or lipopolysaccharides .
as shown in figure  <dig>  the activation of nf-κb increased over 58-fold in hek-293 t cells that were transiently transfected with plasmid encoding gtlr2- <dig> and stimulated with malp- <dig> for 6 hours., however, this phenomenon was not seen with lps stimulation.
in addition, transfection with gtlr2-1–encoding plasmid resulted in a detectable amount activation of nf-κb  in the absence of malp- <dig> stimulation, suggesting that ectopic expression of gtlr <dig> in this human cell line, can mediate a basal level of activation.figure  <dig> expression of goose tlr2- <dig> in hek <dig> cells results in nf-κb activation after stimulation of cells with malp- <dig> or lps.
cells were transfected with nf-κb firefly luciferase reporter and normalizing plasmid alone or with constructs expressing goose tlr2- <dig>  after 16 hours, medium was replaced either with fresh medium alone or medium containing malp- <dig>  at 50 ng/ml or lps at 20 ng/ml.
nf- κb activation levels were measured after six hours by a dual luciferase reporter assay.
the data are presented as mean +/− sd, and statistical analysised by one-way anova.
mg and se infection induced the upregulation of tlr2- <dig> in vivo
exposure to malp- <dig> significantly increased the expression of tlr2- <dig> in pbmcs at 8 h and 12 h post-exposure , with the peak value attained at 8 h after the end of stimulation , while il- <dig> transcription showed a much lee significant increase when compared with tlr2- <dig> figure  <dig> expression of goose tlr2- <dig> and il-1β in pbmcs after stimulation by malp- <dig>  goose pbmcs were isolated and cultured with rpmi  <dig>  after cells were stimulated for 30 min by 50 ng/ml of malp- <dig>  the expression of tlr2- <dig> or il-1β were measured at  <dig>   <dig>   <dig>   <dig> and 12 h after the end of the exposure.
the data are presented as the mean ± sd, and statistically analyzed by one-way anova.
“*” indicates that p ≤  <dig>  for each test.
mg infection significantly upregulated the expression of tlr2- <dig> in the cecum at days  <dig> and  <dig>  in the spleen gland on day  <dig> and in the kidneys on day  <dig> .
furthermore, se infection increased the expression of tlr2- <dig> in the cecum and spleen gland on day  <dig> , with approximately 3-fold increase in both tissues .
interestingly, a downregulation of tlr2- <dig> was also observed in the kidney on both days  <dig> and  <dig> .figure  <dig> expression of goose tlr2- <dig> in indicated tissues after infection with mycoplasma gallisepticum
salmonella enteritidis
and phosphate buffered saline  as a mock.
4-week-old geese were infected with  <dig>  ml mycoplasma gallisepticum  and  <dig>  ml salmonella enteritidis  with intrapleural or intramuscular injections, respectively.
the expression of tlr2- <dig> mrna in the cecum, spleen gland and kidneys of infected geese were detected by qrt-pcr.
the data are presented as the mean ± sd, and statistically analyzed by one-way anova.
“*” and “*” indicate that p ≤  <dig>  and p ≤  <dig>  for each test, respectively.
discussion
the role of tlrs in the immune response to pathogenic bacteria and viruses have been studied intensively possibly due to their ability to recognize pathogens and initiate development of an immune response.
currently,  <dig> tlrs in humans and  <dig> tlrs in chicken have been described but less is known about tlrs in geese.
in the present study, we for the first time cloned and sequenced tlr2- <dig> cdna in goose, and found that the full-length tlr2- <dig> cdna  contained a 5′ untranslated region , an open reading frame  encoding  <dig> amino acids and a 3′ untranslated region .
the sequence homologies of nucleotides and amino acids were  <dig> %,  <dig> %,  <dig>  and  <dig> % respectively in comparison with human, pig, turkey and duck, and the amino acid sequence was  <dig> % homologous with chicken tlr2- <dig> .
we also found that tlr2- <dig> played an role in the recognition of mycoplasma fermentans lipopeptide, mg, se, and induced the activation of nf-κb.table  <dig>
comparison of gtlr2- <dig> amino acid sequence homology with chicken’s tlrs
a
species
gtlr2-1
chtlr1la
chtlr1lb
chtlr2a
chtlr2b
chtlr3
chtlr4
chtlr5
chtlr7
chtlr15
chtlr16
chtlr21
ahomology of goose tlr2- <dig> with tlr <dig> of various species at the nucleotide level.
goose  tlr2- <dig> nucleotide sequence were compared with those of chicken  tlr1la , tlr1lb , tlr2a , tlr2b , tlr <dig> , tlr <dig> , tlr <dig> , tlr <dig> , tlr <dig> , tlr <dig>  and tlr <dig> .
comparison of tlr2- <dig> amino acid sequence homology between various species
a
species
goose
chicken
turkey
bird
duck
human
mouse
pig
cattle
fish
a homology of goose tlr2- <dig> with tlr <dig> of various species at the amino acid level.
goose tlr2- <dig> mrna and encoded amino acid were compared with those of chicken , turkey , zebra finch , duck , human , pig , cattle , mouse  and takifugu rubripes .
the organization of lrrs provides the protein structures for specific interaction with the respective pamp and varies between tlr groups and species.
the ectodomains of tlrs comprise 19– <dig> tandem copies of lrrs, which are thought to be involved in the recognition of various microbial ligands  <cit> .
in contrast, the cytoplasmic portion of tlrs, the tir domain, which has homology with the cytoplasmic domain of human il- <dig>  is the most conserved between the tlrs and has a very ancient evolutionary origin  <cit> .
the tir domain is also involved in bimolecular interactions with myd <dig>  an adaptor protein necessary for signal transduction  <cit> .
based on the predicted amino acid sequence, gtlr2- <dig> consists of an extracellular scaffold of lrr domains and the intracellular tir domain.
variability in amino acid sequences is present in the n-terminal region, resulting in  <dig> lrr domains, as in the chicken.
sequence analysis in goose indicated high conservation of the three subsections of the tir domain  known to be important in signaling  and receptor localization   <cit> .
overall, the gtlr2- <dig> proteins share  <dig> % identity with chicken.
tlr <dig> mrna expression has been reported to be relatively moderate in circulating leukocytes and the spleen, moderate in the ovary, lung, pancreas and placenta, and low in brain, colon, heart, kidney, liver, prostate, small intestine, testis, skeletal muscle and thymus in humans  <cit> .
the pattern of gtlr2- <dig> mrna expression was similar to that in humans according our study.
it is notable that gtlr2- <dig> mrna was higher in skin, intestine and immune tissue, which are all exposed to the external environment.
we hypothesize that this characteristic expression pattern of gtlr2- <dig> is associated with the habitat of the goose, and that frequent contact with microorganisms in the water may increase the expression of tlrs due to frequent challenge by pathogenic bacteria.
however, further study is needed to analyze the expression characteristics of tlrs in skin exposed to water containing different bacterial loads.
in addition to tlr <dig>  tlr <dig> expression has also been reported in a wide range of tissues and is responsible for recognition of pamps including lps of gram-negative bacteria  <cit> .
each of these receptors plays an important role in defense against microbial infection.
tlr2-deficient macrophages have been shown to be hyporesponsive to staphylococcus aureus peptidoglycan, and c3h/hej mice with missense mutations of the tlr <dig> gene were highly susceptible to gram-negative bacterial infection  <cit> .
therefore, it has been suggested that cooperation between tlr <dig> and tlr <dig> is important in the detection of microorganisms and in inducing inflammatory responses.
however, canine tlr <dig> mrna was detected in skin in the absence of tlr <dig> mrna expression  <cit> , and similar findings were reported in human keratinocytes  <cit> .
in our study, goose tlr2- <dig> mrna expression was higher in skin and muscle, which form a first line defense against a variety of pathogens, especially gram-positive bacteria.
in future study, we will explore the expression profile of tlr <dig> and its interaction with tlr <dig> in the skin of the goose.
tlr <dig> acts as a pattern recognition receptor and signals the presence of various bacteria through the activation of nf-κb, not only in human and mouse, but also in chicken  <cit> .
since the tir domain of gtlr2- <dig> was highly homologous to that of human, as well as to oviparous vertebrates, and the amino acid residues responsible for signaling were also apparently conserved, we presumed that goose tlr2- <dig> may be analogues of mammalian tlr <dig>  we therefore tested whether mycoplasma components can activate nf-κb via the tlr pathway.
lps and malp- <dig>  the traditional representative ligands of tlr <dig> and tlr <dig> in mammals  <cit> , were used in the present study.
we found that malp- <dig> but not e. coli lps served as a ligand for nf-κb activation in hek <dig> cells expressing gtlr2- <dig>  indicating that gtlr2- <dig> was an analogue of mammalian tlr <dig> and plays an role in the recognition of malp- <dig>
mycoplasmas were first isolated from geese by kosova and djurisic  <cit> , and further studies also isolated mycoplasmas successfully from geese in association with various conditions such as airsacculitis and peritonitis  <cit> .
intratracheal infection of goslings or goose embryos with mycoplasma and acholeplasma strains resulted in airsacculitis  <cit> .
other studies have shown that mycoplasma cloacae and mycoplasma anseris induced a fall in egg production and increased embryo mortality  <cit> .
the present results found that not only mg, but se infections, induced the expression of tlr2- <dig> in goose in a tissue- and time-dependent manner, which suggested that the immune system of the goose may recognize the pamp of mg and se via tlr2- <dig>  but the fold changes in se challenge geese was smaller than the case of mg,may due to the pamps of se generally accepted to include lps, flagellin, cpg-odn, which may not be the major ligands of tlr <dig> in mammals and chickens.
as mentioned above, the dimerization of tlrs is important in the recognition of pathogens.
synthetic malp- <dig> induces the activation of nf-κb in hek- <dig> cells that are co-transfected with chtlr2a and b  <cit> , and its exogenous agonist does not induce the activation of nf-κb when transfected cells express chtlr2a, b, or chtlr1la or b separately  <cit> .
however, the reverse was observed when expression of chtlr2b/chtlr1la was combined, and in this case activation was induced both by synthetic malp- <dig> and pam <dig>  thus, although there are anomalies between these studies, they do demonstrate that chtlr1ls interact with chtlr2s and can recognize agonists identical to those of the mammalian dimers of tlr <dig> with tlrs <dig>   <dig> and  <dig>  however in geese, further study is needed to explore the interaction between tlrs and the signal pathways are activated by pathogen invasion in particular.
CONCLUSIONS
we cloned goose tlr2- <dig>  examined its expression pattern, investigated the gtlr2-1–mediated signal pathway and the expression of gtlr2- <dig> after pathogenic infection.
these results suggest that gtlr2- <dig> plays a role in the recognition of pathogen-associated molecular patterns in bacteria, and in particular mycoplasma spp.
infections.

 toll-like receptors have emerged as a major component of the vertebrate pattern recognition receptor repertoire, and can recognize conserved pathogen-associated molecular patterns of bacteria, fungi, viruses and parasites as well as some self-ligands, such as host dna in immune complexes and the malarial pigment hemozoin,, but not commensal microbes. upon activation by an agonist, tlrs induce the expression of a wide range of immunoregulatory and effector molecules, which take part in the regulation of immune cell maturation and the clearance of pathogenic microorganisms. in humans and mice,four tlrs are responsible for recognition of antigens from salmonella enteritidis,a gram-negative bacteria, as well as via tlr tlr and tlr2- in chicken.furthermore, tlr dimerizes with tlr or tlr enabling it to recognize microbial triacyl lipoproteins or diacyl lipopeptides found in mycoplasma, lipoteichoic acid of gram-positive bacteria or zymosan of yeasts.however, caution is required in the interpretation of these findings as tlr may not recognize some agonists at physiologically relevant concentrations. recently, human tlr has also been shown to dimerize with tlr enabling activation by triacyl lipoproteins. chicken chtlr1la and b, and chtlr2a and b have been cloned and expressed in mammalian cells by several groups and were demonstrated to be surface- exposed. it was thought that some diseases, such as newcastle disease and h5n avian influenza, occurred only in domestic avian species, did not appear or had only very low morbidity in waterfowl. however, since serotype i avian paramyxoviruses have caused high morbidity and mortality in geese during outbreaks in many provinces of china. interestingly, recent studies have not determined any obvious variation in the gene sequences of apmv-. tlr genes has been identified in mammalian and nonmammalian vertebrates including chicken. however, it is not known whether geese also carry a homologue of mammalian tlr for recognizing the pathogens. in the present study, we cloned and sequenced the goose tlr2- analyzed its expression in several tissues and verified its lipopeptide-mediated activation in hek-293 t cells. meanwhile, the expression of tlr2- in goose tissues after they were challenged with mycoplasma gallisepticum or salmonella enteritidis were also studied.  cloning of goose tlr2-1 r fragments of various sizes were subcloned into the vector pmd18-t, and their sequences were determined. one of the clones was found to contain a sequence similar to the tir domain of chicken tlr2s. a fragment of  bp was obtained by 3′-race-pcr. a 930-bp fragment was then amplified by 5′-race-pcr. a  bp nucleotide sequence representing the full-length, complete cdna of the goose tlr2- gene was obtained from overlapping cdna fragments. the full-length cdna harbored an orf of  bp, encoding a protein of amino acids in length. we aligned the determined nucleotide sequence from geese with sequences in genebank by blast search tool of national center for biotechnology information, and named the present gene as gtlr2- the sequence was submitted to genbank and assigned the accession number genbank: jn the untranslated regions were 359 bp and 378 bp for the 5′-utr and 3′-utr, respectively.figure the results of goose tlr2- gene cloning and eukaryotic expression. electro-phoresis of rt-pcr and race-pcr products of the goose tlr2- gene. lane represents the 5′-race-pcr product, a 930-bp fragment. lane represents the 3′-race-pcr product, a  bp fragment. lane represents the homologous cdna fragments from chicken tlr2- forming a band of 2150 bp. electrophoresis of the full-length tlr2-1encoding an open read frame, a fragment of  bp. immunoblotting of gtlr2- expressed in hek cells, showing a 103-kda band. prediction of protein domains revealed that the putative amino acid sequence consisted of a signal peptide sequence encompassing the first amino acid residues of the n-terminal region, nine leucine-rich repeat domains, a leucine-rich repeat c-terminal domain, a transmembrane domain and a 144-amino-acid toll-interleukin-receptor domain at positions 649– of the carboxy-terminus. highly conserved three subsections of the tir domain is shown in figure  figure nucleotide and deduced amino acid sequence of goose tlr2- complete sequence of the full-length gtlr2- was deposited in the cdna library. translated amino acid sequence is shown under the nucleotide sequence. numbers to the left of each row refer to nucleotides. the cleavage site for the putative signal peptide is indicated by an arrow. leucine-rich repeat domains are shaded in grey. potential n-glycosylation sites are shown as square boxes. the initiation codon is underlined. the predicted transmembrane segment is double underlined. the translational stop site is indicated by an asterisk. the cysteines critical for the maintenance of the structure of leucine-rich repeat c-terminal are marked with coarse underlines. lrr-typ motif is shown by wave lines. tir motif is shown by a long box. tissue distribution analyzed by quantitative rt-pcr the expression of gtlr2- in geese organ tissues was analyzed by quantitative rt-pcr. the level of gtlr2- mrna was highest in kidney, and moderate expression levels were seen in bursa, intestine, brain, stomach and heart. the expression levels in the spleen and lung were relatively low.figure relative gtlr2- expression levels in goose tissues. the expression of the gtlr2- gene in different tissues was analyzed by quantitative rt-pcr. the data are presented as mean +/− sd, and were normalized using the β-actin gene and calculated by the delta ct method. we compared the gtlr2- expression in indicated tissues to that in spleen. “*” and “**”indicates that p ≤  and p ≤  for each test respectively. malp- stimulation of hek- cells expressing goose tlr2- results in nf-κb activation goose tlr2- was cloned downstream from a pcmv promoter with a his-tag fused at the carboxyl terminus. expression of gtlr2- was confirmed by western blot analysis of hek- cells transiently transfected with the gtlr2- expression plasmid, showing a protein band with an expected size of kd. as the activation of tlrs results in its interaction with adaptor molecules and downstream signaling, leading to upregulation of genes with nf-κb elements, a nf-κb luciferase reporter system was used to determine whether the expressed recombinant goose tlr could be activated by exogenous macrophage-activating lipopeptide or lipopolysaccharides. as shown in figure  the activation of nf-κb increased over 58-fold in hek-293 t cells that were transiently transfected with plasmid encoding gtlr2- and stimulated with malp- for 6 hours., however, this phenomenon was not seen with lps stimulation. in addition, transfection with gtlr2-1–encoding plasmid resulted in a detectable amount activation of nf-κb in the absence of malp- stimulation, suggesting that ectopic expression of gtlr in this human cell line, can mediate a basal level of activation.figure expression of goose tlr2- in hek cells results in nf-κb activation after stimulation of cells with malp- or lps. cells were transfected with nf-κb firefly luciferase reporter and normalizing plasmid alone or with constructs expressing goose tlr2- after 16 hours, medium was replaced either with fresh medium alone or medium containing malp- at 50 ng/ml or lps at 20 ng/ml. nf- κb activation levels were measured after six hours by a dual luciferase reporter assay. the data are presented as mean +/− sd, and statistical analysised by one-way anova. mg and se infection induced the upregulation of tlr2- in vivo exposure to malp- significantly increased the expression of tlr2- in pbmcs at 8 h and 12 h post-exposure, with the peak value attained at 8 h after the end of stimulation, while il- transcription showed a much lee significant increase when compared with tlr2- figure expression of goose tlr2- and il-1β in pbmcs after stimulation by malp- goose pbmcs were isolated and cultured with rpmi after cells were stimulated for 30 min by 50 ng/ml of malp- the expression of tlr2- or il-1β were measured at and 12 h after the end of the exposure. the data are presented as the mean ± sd, and statistically analyzed by one-way anova. “*” indicates that p ≤  for each test. mg infection significantly upregulated the expression of tlr2- in the cecum at days and in the spleen gland on day and in the kidneys on day. furthermore, se infection increased the expression of tlr2- in the cecum and spleen gland on day, with approximately 3-fold increase in both tissues. interestingly, a downregulation of tlr2- was also observed in the kidney on both days and.figure expression of goose tlr2- in indicated tissues after infection with mycoplasma gallisepticum salmonella enteritidis and phosphate buffered saline as a mock. 4-week-old geese were infected with  ml mycoplasma gallisepticum and  ml salmonella enteritidis with intrapleural or intramuscular injections, respectively. the expression of tlr2- mrna in the cecum, spleen gland and kidneys of infected geese were detected by qrt-pcr. the data are presented as the mean ± sd, and statistically analyzed by one-way anova. “*” and “*” indicate that p ≤  and p ≤  for each test, respectively. discussion the role of tlrs in the immune response to pathogenic bacteria and viruses have been studied intensively possibly due to their ability to recognize pathogens and initiate development of an immune response. currently, tlrs in humans and tlrs in chicken have been described but less is known about tlrs in geese. in the present study, we for the first time cloned and sequenced tlr2- cdna in goose, and found that the full-length tlr2- cdna contained a 5′ untranslated region, an open reading frame encoding amino acids and a 3′ untranslated region. the sequence homologies of nucleotides and amino acids were %, %, and % respectively in comparison with human, pig, turkey and duck, and the amino acid sequence was % homologous with chicken tlr2-. we also found that tlr2- played an role in the recognition of mycoplasma fermentans lipopeptide, mg, se, and induced the activation of nf-κb.table comparison of gtlr2- amino acid sequence homology with chicken’s tlrs a species gtlr2-1 chtlr1la chtlr1lb chtlr2a chtlr2b chtlr3 chtlr4 chtlr5 chtlr7 chtlr15 chtlr16 chtlr21 ahomology of goose tlr2- with tlr of various species at the nucleotide level. goose tlr2- nucleotide sequence were compared with those of chicken tlr1la, tlr1lb, tlr2a, tlr2b, tlr, tlr, tlr, tlr, tlr, tlr and tlr. comparison of tlr2- amino acid sequence homology between various species a species goose chicken turkey bird duck human mouse pig cattle fish a homology of goose tlr2- with tlr of various species at the amino acid level. goose tlr2- mrna and encoded amino acid were compared with those of chicken, turkey, zebra finch, duck, human, pig, cattle, mouse and takifugu rubripes. the organization of lrrs provides the protein structures for specific interaction with the respective pamp and varies between tlr groups and species. the ectodomains of tlrs comprise 19– tandem copies of lrrs, which are thought to be involved in the recognition of various microbial ligands. in contrast, the cytoplasmic portion of tlrs, the tir domain, which has homology with the cytoplasmic domain of human il- is the most conserved between the tlrs and has a very ancient evolutionary origin. the tir domain is also involved in bimolecular interactions with myd an adaptor protein necessary for signal transduction. based on the predicted amino acid sequence, gtlr2- consists of an extracellular scaffold of lrr domains and the intracellular tir domain. variability in amino acid sequences is present in the n-terminal region, resulting in lrr domains, as in the chicken. sequence analysis in goose indicated high conservation of the three subsections of the tir domain known to be important in signaling and receptor localization. overall, the gtlr2- proteins share % identity with chicken. tlr mrna expression has been reported to be relatively moderate in circulating leukocytes and the spleen, moderate in the ovary, lung, pancreas and placenta, and low in brain, colon, heart, kidney, liver, prostate, small intestine, testis, skeletal muscle and thymus in humans. the pattern of gtlr2- mrna expression was similar to that in humans according our study. it is notable that gtlr2- mrna was higher in skin, intestine and immune tissue, which are all exposed to the external environment. we hypothesize that this characteristic expression pattern of gtlr2- is associated with the habitat of the goose, and that frequent contact with microorganisms in the water may increase the expression of tlrs due to frequent challenge by pathogenic bacteria. however, further study is needed to analyze the expression characteristics of tlrs in skin exposed to water containing different bacterial loads. in addition to tlr tlr expression has also been reported in a wide range of tissues and is responsible for recognition of pamps including lps of gram-negative bacteria. each of these receptors plays an important role in defense against microbial infection. tlr2-deficient macrophages have been shown to be hyporesponsive to staphylococcus aureus peptidoglycan, and c3h/hej mice with missense mutations of the tlr gene were highly susceptible to gram-negative bacterial infection. therefore, it has been suggested that cooperation between tlr and tlr is important in the detection of microorganisms and in inducing inflammatory responses. however, canine tlr mrna was detected in skin in the absence of tlr mrna expression, and similar findings were reported in human keratinocytes. in our study, goose tlr2- mrna expression was higher in skin and muscle, which form a first line defense against a variety of pathogens, especially gram-positive bacteria. in future study, we will explore the expression profile of tlr and its interaction with tlr in the skin of the goose. tlr acts as a pattern recognition receptor and signals the presence of various bacteria through the activation of nf-κb, not only in human and mouse, but also in chicken. since the tir domain of gtlr2- was highly homologous to that of human, as well as to oviparous vertebrates, and the amino acid residues responsible for signaling were also apparently conserved, we presumed that goose tlr2- may be analogues of mammalian tlr we therefore tested whether mycoplasma components can activate nf-κb via the tlr pathway. lps and malp- the traditional representative ligands of tlr and tlr in mammals, were used in the present study. we found that malp- but not e. coli lps served as a ligand for nf-κb activation in hek cells expressing gtlr2- indicating that gtlr2- was an analogue of mammalian tlr and plays an role in the recognition of malp- mycoplasmas were first isolated from geese by kosova and djurisic, and further studies also isolated mycoplasmas successfully from geese in association with various conditions such as airsacculitis and peritonitis. intratracheal infection of goslings or goose embryos with mycoplasma and acholeplasma strains resulted in airsacculitis. other studies have shown that mycoplasma cloacae and mycoplasma anseris induced a fall in egg production and increased embryo mortality. the present results found that not only mg, but se infections, induced the expression of tlr2- in goose in a tissue- and time-dependent manner, which suggested that the immune system of the goose may recognize the pamp of mg and se via tlr2- but the fold changes in se challenge geese was smaller than the case of mg,may due to the pamps of se generally accepted to include lps, flagellin, cpg-odn, which may not be the major ligands of tlr in mammals and chickens. as mentioned above, the dimerization of tlrs is important in the recognition of pathogens. synthetic malp- induces the activation of nf-κb in hek- cells that are co-transfected with chtlr2a and b, and its exogenous agonist does not induce the activation of nf-κb when transfected cells express chtlr2a, b, or chtlr1la or b separately. however, the reverse was observed when expression of chtlr2b/chtlr1la was combined, and in this case activation was induced both by synthetic malp- and pam thus, although there are anomalies between these studies, they do demonstrate that chtlr1ls interact with chtlr2s and can recognize agonists identical to those of the mammalian dimers of tlr with tlrs and however in geese, further study is needed to explore the interaction between tlrs and the signal pathways are activated by pathogen invasion in particular.  we cloned goose tlr2- examined its expression pattern, investigated the gtlr2-1–mediated signal pathway and the expression of gtlr2- after pathogenic infection. these results suggest that gtlr2- plays a role in the recognition of pathogen-associated molecular patterns in bacteria, and in particular mycoplasma spp. infections. 
geese tlr2- is a functional homologue of tlr present in other species and plays an important role in bacterial recognition in geese. in this study, we cloned the goose tlr2- gene using rapid amplification of cdna ends and showed that geese tlr2- encoded a 793-amino-acid protein, containing a signal secretion peptide, an extracellular leucine-rich repeat domain, a transmembrane domain and a toll/interleukin- receptor signaling domain deduced from amino acid sequence. furthermore, geese infected with mycoplasma gallisepticum and salmonella enteritidis showed significant upregulation of tlr2- in both in vivo and in vitro. hek cells transfected with plasmids carrying goose tlr2- and nf-κb-luciferase responded significantly to stimulation with mycoplasma fermentans lipopeptide.
 toll-like receptor, an important pattern recognition receptor, activates proinflammatory pathways in response to various pathogens. it has been reported in humans and chicken, but not in geese, an important waterfowl species in china. since some vaccines stimulate robust immune responsesl in chicken but not in geeeses we speculated that their immune systems are different.  in this study, we cloned the goose tlr2- gene using rapid amplification of cdna ends and showed that geese tlr2- encoded a 793-amino-acid protein, containing a signal secretion peptide, an extracellular leucine-rich repeat domain, a transmembrane domain and a toll/interleukin- receptor signaling domain deduced from amino acid sequence. tlr2- shared %– % homology with its homologues in other species. tissue expression of geese tlr2- varied markedly, and was higher in kidney, cloacal bursa, skin and brain compared to other organs/tissues. hek cells transfected with plasmids carrying goose tlr2- and nf-κb-luciferase responded significantly to stimulation with mycoplasma fermentans lipopeptide. furthermore, geese infected with mycoplasma gallisepticum and salmonella enteritidis showed significant upregulation of tlr2- in both in vivo and in vitro.  geese tlr2- is a functional homologue of tlr present in other species and plays an important role in bacterial recognition in geese. keywords goosetlr2-1nf-кbmycoplasmasalmonellainfectionissue-copyright-statement© the author 2015 