Spreading of<Emphasis Type="Italic">Salmonella enteritidis</Emphasis> in the cecum of chickens

Adhesion and colonization of high (2 x 10(8) CFU) and low doses (2 x 10(2) CFU) of Salmonella enteritidis (phage type 4) was determined in the ceca collected 6 h-4 weeks after inoculation (pi), of 1-d-old White Plymouth Rock orally-inoculated chickens. S. enteritidis was associated with the epithelial surface of the villi in the low-dose group 18 h-7 d pi, the penetration in the cecal lamina propria was observed on day 1 and 10 pi. In the high-dose group, adhesion and colonization was observed in all birds killed 6 h-14 d pi; penetration of the bacteria into the cecal lamina propria was seen 1-21 d pi. Large numbers of macrophage-like cells containing S. enteritidis were observed in the cecal lamina propria on days 3-21 pi. Colonization and migration by S. enteritidis in the intestinal tract of chickens was shown to be dose dependent.     

Differential mRNA expression of the avian-specific toll-like receptor 15 between heterophils from <Emphasis Type="Italic">Salmonella</Emphasis>-susceptible and -resistant chickens

Pattern recognition receptors (PRRs) are essential for recognition of conserved molecular constituents found on infectious microbes. Toll-like receptors (TLRs) are a critical component of the PRR repertoire and are coupled to downstream production of cytokines, chemokines, and antimicrobial peptides by TLR adaptor proteins. Our laboratory previously demonstrated a role for TLR function in the differential innate response of two lines of chickens to bacterial infections. The aim of the present study was to elucidate the role of TLRs in the differential innate responsiveness by measuring differences between lines A (resistant) and B (susceptible) in heterophil mRNA expression of selected TLRs (TLRs 4, 5, and 15) and TLR adaptor proteins (MyD88, TRIF, and TIRAP) in response to stimulation with Salmonella enterica serovar Enteritidis (SE). Although heterophils from both lines had significantly increased expression of TLR 15 mRNA in response to stimulation with SE, heterophils from chickens resistant to infection with SE had significantly greater levels of TLR 15 mRNA expression prior to and following stimulation with SE than heterophils from chickens susceptible to infection with SE. No significant differences were noted between lines in nonstimulated levels of TIRAP, but upon SE stimulation, line A birds had higher levels of expression than B birds. No significant differences were found in heterophils between lines for mRNA expression of TLRs 4 and 5 nor MyD88 and TRIF. These data indicate that differences in the gene expression of TLR 15 by heterophils likely accounts for some of the observed differences between the lines in their susceptibility to infection.     

重组SEF21脂质体口服疫苗对肠炎沙门菌感染的作用

目的探讨制备脂质体包裹重组SEF21疫苗,并评价其在预防肠炎沙门菌(S.enteritidis)感染中的作用。方法利用PCR获得SEF21基因,并连接至pET-28a(+)载体。将pET-28a(+)-SEF21在BL21(DE3)大肠埃希菌中表达,通过镍层析柱纯化高表达的rSEF21蛋白。制备脂质体包裹rSEF21疫苗,并对鸡进行2次免疫,然后利用S.enteritidis进行攻毒实验。ELISA检测血清以及肠内容物中的抗体效价。结果所有被免疫鸡的血清及肠黏液中产生了高效价的IgG和IgA抗体。脂质体包裹rSEF21所免疫的鸡的粪便样本中S.enteritidis数量明显下降。结论口服脂质体包裹的重组SEF21蛋白疫苗能有效保护鸡对抗S.enteritidis感染。     

Expression profiles of Toll-like receptors 1, 2 and 5 in selected organs of commercial and indigenous chickens

Toll-like receptors (TLRs) are members of the cellular receptors that constitute a major component of the evolutionary conserved pattern recognition system (PRR). TLRs are expressed in a wide variety of tissues and cell types. In this study we compared the expression profiles of the chicken TLR1, TLR2 and TLR5 genes in a range of organs (lung, ovary, liver, thymus, duodenum, spleen and large intestine) in commercial Hy-Line (HL) and indigenous Green-legged Partridgelike (GP) chickens. The level of mRNA was determined with RT-qPCR using the TaqMan probes for target and reference (ACTB) genes. We determined that the tissue profiles differed with respect to each TLR and they were ranked as follows: spleen, lungs, large intestine (TLR1), large intestine, lungs, thymus/ovary (TLR2) and lungs, thymus, liver (TLR5). A differential expression between HL and GP chickens was determined for TLR1 and TLR5 genes in large intestine and thymus of HL (P?<?0.05) and GP (P?<?0.05) chickens. We conclude that the commercial chickens expressed higher levels of TLR1 mRNA in large intestine and TLR5 mRNA in thymus than indigenous chickens.     

Heterophils are associated with resistance to systemic Salmonella enteritidis infections in genetically distinct chicken lines

Heterophils mediate acute protection against Salmonella in young poultry. We evaluated susceptibility of genetically distinct lines of broilers to systemic Salmonella enteritidis (SE) infections. SE was administered into the abdomen of day-old chickens (parental lines [A and B]; F1 reciprocal crosses [C and D]) to assess modulation of leukocytes and survivability of chickens. Line A was more resistant to SE than line B; likewise cross D was more resistant than cross C. Significantly more heterophils migrated to the abdominal cavity post-infection in the resistant lines. These data indicate that increased heterophil influx to the infection site contributes to increased resistance against systemic SE infections in neonatal chickens.     

TLR Ligands Induce Antiviral Responses in Chicken Macrophages

Chicken macrophages express several receptors for recognition of pathogens, including Toll-like receptors (TLRs). TLRs bind to pathogen-associated molecular patterns (PAMPs) derived from bacterial or viral pathogens leading to the activation of macrophages. Macrophages play a critical role in immunity against viruses, including influenza viruses. The present study was designed to test the hypothesis that treatment of chicken macrophages with TLR ligands reduces avian influenza replication. Furthermore, we sought to study the expression of some of the key mediators involved in the TLR-mediated antiviral responses of macrophages. Chicken macrophages were treated with the TLR2, 3, 4, 7 and 21 ligands, Pam3CSK4, poly(I:C), LPS, R848 and CpG ODN, respectively, at different doses and time points pre- and post-H4N6 avian influenza virus (AIV) infection. The results revealed that pre-treatment of macrophages with Pam3CSK4, LPS and CpG ODN reduced the replication of AIV in chicken macrophages. In addition, the relative expression of genes involved in inflammatory and antiviral responses were quantified at 3, 8 and 18 hours post-treatment with the TLR2, 4 and 21 ligands. Pam3CSK4, LPS and CpG ODN increased the expression of interleukin (IL)-1β, interferon (IFN)-γ, IFN-β and interferon regulatory factor (IFR) 7. The expression of these genes correlated with the reduction of viral replication in macrophages. These results shed light on the process of immunity to AIV in chickens.     

Histone Methylation Analysis and Pathway Predictions in Chickens after MDV Infection

Marek's disease (MD) is a lymphoproliferative disease in chicken induced by Marek's disease virus (MDV). Although studies have focused on the genetic differences between the resistant and susceptible chicken, less is known about the role of epigenetic factors in MD. In this study, genome-wide histone modifications in the non-MHC-associated resistant and susceptible chicken lines were examined. We found that tri-methylation at histone H3 Lys4 (H3K4me3) enrichment is positively correlated with the expression of protein coding genes as well as microRNA (miRNA) genes, whereas tri-methylation at histone H3 Lys27 (H3K27me3) exhibits a negative correlation. By identifying line-specific histone modifications in MDV infection, we found unique H3K4me3 islands in the resistant chicken activated genes, which are related to immune response and cell adhesion. Interestingly, we also found some miRNAs from unique H3K27me3 patterns in the susceptible chickens that targeted genes involved in 5-hydroxytryptamine (5-HT)-receptor and adrenergic receptor pathways. In conclusion, dynamic line-specific histone modifications in response to MDV infection suggested that intrinsic epigenetic mechanisms may play a role in MD-resistance and -susceptibility.     

Changes in the expression of Toll-like receptors in the chicken testis during sexual maturation and Salmonella infection

Rooster infertility is a major concern in the poultry industry and chicken male reproductive organs are the infectious tissues of various pathogenic microorganisms. Protection of the chicken male reproductive organs from pathogens is therefore an essential aspect of reproductive physiology. Recently Toll-like receptors (TLRs) have been identified as one of the key components of innate immunity in vertebrate species and have been reported to be expressed in the reproductive organs in various female species, including the chicken. However, mechanisms of antimicrobial protection of male reproductive organs mediated by TLRs are poorly understood. The objectives of this study were to determine the expression profile of the entire family of the ten chicken TLR genes in the chicken testis, to investigate whether sexual maturation affects their testicular mRNA abundance and to determine the changes in their expression levels in response to Salmonella enteritidis (SE) infection. RNA was extracted from the testis of healthy pre-pubertal, sexually mature and aged birds, and from sexually mature SE infected birds. RT-PCR analysis revealed that all TLRs, apart from TLR1-1 (TLR6), were expressed in the chicken testis. Quantitative real-time PCR analysis revealed that the testicular mRNA abundance of certain TLRs was developmentally regulated with respect to sexual maturation, while SE infection resulted in a significant induction of TLR2-1, 4, 5, 15 and 21 in the testis of sexually mature birds compared, to healthy birds of the same age. These findings provide strong evidence to suggest a key role of TLRs in the innate immune responses of chicken testis against Salmonella colonization.     

Correlation of acute with chronic hypoxic pulmonary hypertension in cattle.

We investigated acute and chronic hypoxic pulmonary pressor responses in two groups of calves, one bred to be susceptible, the other resistant to high-altitude pulmonary hypertension. Twelve 5-mo-old susceptible calves residing at 1,524 m increased their mean pulmonary arterial pressure from 26 +/- 2 (SE) to 55 +/- 4 mmHg during 2 h at a simulated altitude of 4,572 m. In 10 resistant calves pressure increased from 22 +/- 1 to 37 +/- 2 mmHg. Five calves were selected from each group for further study. When 9 mo old, the 5 susceptible calves again showed a greater pressor response to acute hypoxia (27 +/- 1 to 55 +/- 4 mmHg) than did 5 resistant calves (23 +/- 1 to 41 +/- 3 mmHg). When 12 mo old, the 5 susceptible calves also developed a greater increase in pulmonary arterial pressure (21 +/- 2 to 9 +/- 4 mmHg) during 18 days at 4,572 m than did the 5 resistant calves (21 +/- 1 to 64 +/- 4 mmHg). Acute and chronic hypoxic pulmonary pressor responses were highly correlated (r = 0.91; P less than 0.001) indicating that they were probably produced through a common mechanism.     

Gastrointestinal nematode challenge induces some conserved gene expression changes in the gut mucosa of genetically resistant sheep

Sheep have a varying ability to resist infection with gastrointestinal nematodes. This ability is due in part to genetic differences that exist between individuals. In order to define these differences we have used real-time PCR to quantify gene expression responses in the gut mucosal surface of genetically resistant and susceptible sheep, following a nematode challenge. Expression profiles were determined in response to two different nematode species, Haemonchus contortus and Trichostrongylus colubriformis, and in divergent sheep originating from two different genetic backgrounds. Results show that the response generated differs between resistant and susceptible animals and is further impacted by the origin of the sheep and nematode species used for challenge. However, some conserved features of a response mounted by a resistant or a susceptible animal were identified. Genes found to be more abundantly expressed in resistant animals include markers of an early inflammatory response, several Toll-like receptors (TLR2, 4, 9) and free radical producing genes (DUOX1 and NOS2A). Conversely, genes differentiating susceptible animals indicate a prolonged response and development of a chronic inflammatory state, characterised by elevated expression of members of the NF-kappabeta signalling pathway (IKBKB and NFKBIA) together with delayed expression of regulatory markers such as IL2RA (CD25), IL10 and TGFbeta2. While multiple nematode response pathways were identified, the identification of conserved aspects of the response which associate with resistance provides evidence that alternative nematode control strategies, such as breeding for resistant animals, may be feasible.     

Inhibition of NF-kB 1 (NF-kBp50) by RNA interference in chicken macrophage HD11 cell line challenged with Salmonellaenteritidis

The NF-kB pathway plays an important role in regulating the immunity response in animals. In this study, small interfering RNAs (siRNA) were used to specifically inhibit NF-kB 1 expression and to elucidate the role of NF-kB in the signal transduction pathway of the Salmonella challenge in the chicken HD11 cell line. The cells were transfected with either NF-kB 1 siRNA, glyceraldehyde 3-phosphate dehydrogenase siRNA (positive control) or the negative control siRNA for 24 h, followed by Salmonella enteritidis (SE) challenge or non-challenge for 1 h and 4 h. Eight candidate genes related to the signal pathway of SE challenge were selected to examine the effect of NF-kB 1 inhibition on their expressions by mRNA quantification. The results showed that, with a 36% inhibition of NF-kB 1 expression, gene expression of both Toll-like receptor (TLR) 4 and interleukin (IL)-6 was consistently and significantly increased at both 1 h and 4 h following SE challenge, whereas the gene expression of MyD88 and IL-1β was increased at 1 h and 4 h, respectively. These findings suggest a likely inhibitory regulation by NF-kB 1, and could lay the foundation for studying the gene network of the innate immune response of SE infection in chickens.     

A locus on chromosome 9 is associated with differential response of 129S1/SvImJ and FVB/NJ strains of mice to systemic LPS

Although polymorphisms in TLR receptors and downstream signaling molecules affect the innate immune response, these variants account for only a portion of the ability of the host to respond to microorganisms. To identify novel genes that regulate the host response to systemic lipopolysaccharide (LPS), we created an F2 intercross between susceptible (FVB/NJ) and resistant (129S1/SvImJ) strains, challenged F2 progeny with LPS via intraperitoneal injection, and phenotyped 605 animals for survival and another 500 mice for serum concentrations of IL-1?? and IL-6. Genome-wide scans were performed on pools of susceptible and resistant mice for survival, IL-1??, and IL-6. This approach identified a locus on the telomeric end of the q arm of chromosome 9 (0?C40?Mb) that was associated with the differences in morbidity and serum concentrations of IL-1?? and IL-6 following systemic LPS in FVB/NJ and 129S1/SvImJ strains of mice. Fine mapping narrowed the locus to 3.7?Mb containing 11 known genes, among which are three inflammatory caspases. We studied expression of genes within the locus by quantitative RT-PCR and showed that Casp1 and Casp12 levels are unaffected by LPS in both strains, whereas Casp4 is highly induced by LPS in FVB/NJ but not in 129S1/SvImJ mice. In conclusion, our mapping results indicate that a 3.7-Mb region on chromosome 9 contains a gene that regulates differential response to LPS in 129S1/SvImJ and FVB/NJ strains of mice. Differences in the induction of Casp4 expression by LPS in the two strains suggest that Casp4 is the most likely candidate gene in this region.     

Divergent Toll-like receptors in catfish (Ictalurus punctatus): TLR5S, TLR20, TLR21

Toll-like receptors (TLR) mediate pathogen recognition in vertebrate species through detection of conserved microbial ligands. Families of TLR molecules have been described from the genomes of the teleost fish model species zebrafish and Takifugu, but much research remains to characterize the full length sequences and pathogen specificities of individual TLR members in fish. While the majority of these pathogen receptors are conserved among vertebrate species with clear orthologues present in fish for most mammalian TLRs, several interesting differences are present in the TLR repertoire of teleost fish when compared to that of mammals. A soluble form of TLR5 has been reported from salmonid fish and Takifugu rubripes which is not present in mammals, and a large group of TLRs (arbitrarily numbered 19-23) was identified from teleost genomes with no easily discernible orthologues in mammals. To better understand these teleost adaptations to the TLR family, we have isolated, sequenced, and characterized the full-length cDNA and gene sequences of TLR5S, TLR20, and TLR21 from catfish as well as studied their expression pattern in tissues. We also mapped these genes to bacterial artificial chromosome (BAC) clones for genome analysis. While TLR5S appeared to be common in teleost fish, and TLR21 is common to birds, amphibians and fish, TLR20 has only been identified in zebrafish and catfish. Phylogenetic analysis of catfish TLR20 indicated that it is closely related to murine TLR11 and TLR12, two divergent TLRs about which little is known. All three genes appear to exist in catfish as single copy genes.     

Differential Toll-Like Receptor (TLR) mRNA Expression Patterns during Chicken Embryological Development

Toll-like receptors (TLRs), important components of innate immune response, play a pivotal role in early recognition of pathogen as well as in the initiation of robust and specific adaptive immune response. In the present study, the expression profile of chicken TLRs (TLR2A, TLR3, TLR4, TLR5, TLR7, TLR15, and TLR21) in various chicken embryonic tissues during embryo development was examined by real-time PCR assay. All the TLR mRNAs were expressed in whole embryonic tissue as early as 3rd embryonic day (ED). Four of the seven TLRs (TLR2, TLR3, TLR4, and TLR7) mRNA expressions were significantly (P < 0.01) higher at 12ED relative to expression at 3 ED, whereas TLR15 mRNA expression was significantly (P < 0.01) higher on 7ED and TLR5 and 21 were highly expressed on 18 ED. Among all the TLRs investigated TLR4 mRNA was the highest expressed and TLR15 mRNA expression was the lowest in all tissues during chicken embryo development. Tissue wise analysis of mRNA expression of TLRs showed that liver expressed significantly (P < 0.01) higher levels of most of the genes (TLR2, TLR4, and TLR21). However no significant difference was found in TLR15 mRNA expression among the tissues during development. Our results suggest the innate preparedness of chicken embryos and also a possible role for TLRs in the regulation of chicken embryo development that needs to be further evaluated.     

Differences in mortality, growth, lysozyme, and toll-like receptor gene expression among genetic groups of catfish exposed to virulent Edwardsiella ictaluri

Survivorship to ESC (enteric septicemia of catfish) varies among and within strains of commercially raised catfish, however the immunological basis for differences in susceptibility is not well-understood. We assessed the effect of pathogen challenge with Edwardsiella ictaluri on five genetic groups of catfish by measuring both phenotypic response (mortality, pathogen levels, specific growth rate), and three measures of immune response, including lysozyme activity and mRNA expression of two toll-like receptors (TLR3 and TLR5). Both mortality and pathogen loads, in addition to non-specific immune response, consistently ranged from the least susceptible Blue catfish (24%, 3.4 x 10(2)+/-9.3 x 10(1)cell-equivalents/mg, 13.2+/-3.2U/mL tissue, respectively) to the most susceptible 103 channel catfish (65%, 1.1x10(4)+/-6.4 x 10(3)cell-equivalents/mg tissue, 67.3+/-28.7U/mL, respectively). Similarly, specific growth rate was reduced in exposed fish, compared to non-exposed controls, only in the most susceptible genetic groups (P=0.0051). Trends in mRNA expression levels were apparent in each tissue type for both genes. In kidney, differences were evident in expression of both TLR3 and TLR5 mRNA between strains early and late in challenge (P<0.01). TLR5 mRNA showed significant downregulation in all strains on days 1 and 4 (P=0.0001). In spleen, all strains had elevated levels of TLR3 (P=0.0050) and TLR5 mRNA (P<0.0001) only 1day post-exposure. In stomach, only one strain (103 x RR) showed upregulation (P=0.0063) throughout challenge. The relationship of phenotypic (mortality and growth) and immune responses measured here, suggests that variation in susceptibility to ESC is a function of differences in innate immune response. Understanding these differences will be crucial for enhancing the immune system through selective breeding and in developing disease management protocols for channel catfish.     

Expression analysis of turkey (Meleagris gallopavo) toll-like receptors and molecular characterization of avian specific TLR15

Toll-like receptors (TLRs) constitute a multi-gene family, which plays a pivotal role in sensing invading pathogens by virtue of conserved microbial patterns. TLR repertoire of chicken and zebra finch has been well studied. However TLR family of other avian species is yet to be characterized. In the present study, we identified TLR repertoire of turkey, characterized avian specific receptor TLR15 in turkey and profiled the TLRs expressions in a range of tissues of turkey poults. All ten TLR genes orthologous to chicken TLR repertoire were found in turkey. Turkey TLR genes showed 81-93 % similarity at amino acid level to their chicken counter parts. Phylogenetic analysis confirmed the orthologous relationship of turkey TLRs with chicken and zebra finch TLRs. Open reading frame of turkey TLR15 was 2,607 bp long encoding 868 amino acids similar to that of broiler chicken and showed 92.4, 91.1 and 69.5 % identity at amino acid levels with chicken, Japanese quail and zebra finch TLR15 sequences respectively. Overall TLR expression was highest for TLR4 and lowest for TLR21. TLR1A, 2A, 2B and 21 were significantly higher in liver than other tissues investigated (P < 0.01). TLR3 expression was significantly higher in bone marrow (BM) and spleen in comparison to other tissues studied (P < 0.01). Furthermore, no significant differences in the expression levels of TLR1B, 4, 5, 7 and 15 genes were detected among the tissues studied. Our findings contribute to the characterization of innate immune system of birds and show the innate preparedness of young turkey poults to a range of pathogens.