Molecular characterization of T-cell immunoglobulin mucin domain-3 and Galectin-9 genes of swamp- and riverine-type water buffaloes.

Molecular characterization of T-cell immunoglobulin mucin domain-3 (TIM-3) and Galectin-9 (GAL-9) genes of swamp- and riverine-type water buffaloes was conducted to compare these genes with other species; determine the unique characteristic specific in water buffalo; and provide baseline information for the assessment of disease progression in buffalo species. TIM-3 and GAL-9 genes were amplified, purified, sequenced and characterized. The sequence result of TIM-3 in both types of water buffaloes contained 843 nucleotides encoding to 280 amino acids while GAL-9 of swamp-type and riverine-type water buffaloes contained 1023 and 972 nucleotides encoding to 340 and 323 amino acids, respectively. Meanwhile, the nucleotide and amino sequence of TIM-3 in water buffalo were 83-98% and 94-97% identical with other artiodactyl species, respectively. On the other hand, GAL-9 nucleotide and amino acid sequence in water buffalo were 85-98% and 76-96% identical with other artiodactyl species. The tyrosine-kinase phosphorylation motif and potential glycosylation sites were conserved within the tribe Bovinae. It is imperative to have further studies in the assessment of the role of these genes in disease progression in water buffalo during chronic infection. The study is the first report that describes the genetic characteristic of TIM-3 and GAL-9 genes in water buffalo.

Suppressive effects of neutrophil by Salp16-like salivary gland proteins from Ixodes persulcatus†Schulze tick.

Salp16, a 16-kDa tick salivary gland protein, is known to be the molecule involved in the transmission of Anaplasma phagocytophilum, an obligate intracellular pathogen causing zoonotic anaplasmosis, from its mammalian hosts to Ixodes scapularis. Recently, the presence of A. phagocytophilum was documented in Japan and Ixodes persulcatus was identified as one of its vectors. The purpose of this study was to identify Salp16 genes in I. persulcatus and characterize their function. Two cDNA clones encoding the Salp16-like sequences were obtained from the salivary glands of fed female I. persulcatus ticks and designated Salp16 Iper1 and Iper2. Gene expression analyses showed that the Salp16 Iper genes were expressed specifically in the salivary glands and were up-regulated by blood feeding. These proteins attenuated the oxidative burst of activated bovine neutrophils and inhibited their migration induced by the chemoattractant interleukin-8 (IL-8). These results demonstrate that Salp16 Iper proteins contribute to the establishment of blood feeding as an immunosuppressant of neutrophil, an essential factor in innate host immunity. Further examination of the role of Salp16 Iper in the transmission of pathogens, including A. phagocytophilum, will increase our understanding of the tick-host-pathogen interface.

Kinetics of regulatory dendritic cells in inflammatory responses during Trypanosoma evansi infection.

Trypanosoma evansi (T. evansi) causes a wasting disease in almost all mammals. Trypanosoma evansi infection gives rise to the inflammatory responses that contribute to the development of inflammation-associated tissue injury. To determine what kinds of inflammatory molecules play roles in the pathogenicity of T. evansi infection, polymerase chain reaction array analysis was performed on samples from the infected and uninfected mice. The inflammatory cytokine and chemokine storm, caused mainly by macrophages, was observed. On the other hand, the expression levels of Ccl8 and Il10 in splenocytes were also markedly increased. These results suggested an augmentation in the number and activity of regulatory dendritic cells (DCs). Therefore, the kinetics of regulatory DCs in T. evansi-infected mice were investigated. During T. evansi infection, the regulatory DCs became prevalent, with reducing the amount of inflammatory DCs. Interestingly, when the regulatory DCs were implanted into T. evansi-infected mice, the survival was prolonged, and the expression levels of inflammatory molecules were suppressed. Taken together, these results showed that a subset of regulatory DCs acted as a potential regulator of the inflammatory responses.

Molecular cloning and characterization of Th1 and Th2 cytokines of African buffalo (Syncerus caffer).

The African buffalo (Syncerus caffer) has been implicated as the reservoir of several bovine infectious agents. However, there is insufficient information on the protective immune responses in the African buffalo, particularly in infected animals. In this study, we analysed Th1 cytokines IL-2 and IFN-γ, and Th2 cytokines IL-4 and IL-10. The cloned cDNA of IL-2, IL-4, IL-10 and IFN-γ contained an open reading frame of 468, 501, 408 and 540 nucleotides, encoding polypeptides of 155, 166, 135 and 179 amino acids, respectively. Nucleotide sequence homology of IL-2, IFN-γ and IL-4 was more than 98% between the African buffalo and cattle, which resulted in identical polypeptides. Meanwhile, IL-10 gene of African buffalo and cattle had 95% homology in nucleotide sequence, corresponding to thirteen amino acid residues substitution. Cysteine residues and potential glycosylation sites were conserved within the family Bovinae. Phylogenetic analyses including cytokines of the African buffalo placed them within a cluster comprised mainly of species belonging to the order Artiodactyla, including cattle, water buffalo, sheep, goat, pig and artiodactyl wildlife. A deeper understanding of the structure of these cytokines will shed light on their protective role in the disease-resistant African buffalo in comparison with other closely related species.

Prostaglandin-related immune suppression in cattle.

Prostaglandins (PGs) are lipid mediators derived from arachidonic acid by several enzymes including cyclooxygenase (COX)-1 and COX-2. We have previously shown that PGE2 regulates immune responses, such as Th1 cytokine production and T-cell proliferation, in cattle. However, it is still unclear whether other PGs are involved in the regulation of immune responses in cattle. Here, immunosuppressive profiles of PGs (PGA1, PGB2, PGD2, PGE2, PGF1α and PGF2α) were firstly examined using bovine peripheral blood mononuclear cells (PBMCs). In addition to PGE2, PGA1 significantly inhibited Th1 cytokine production from PBMCs in cattle. Further analyses focusing on PGA1 revealed that treatment with PGA1 in the presence of concanavalin A (con A) downregulated CD69, an activation marker, and IFN-γ expression in both CD4+ and CD8+ T cells. Sorted CD3+ T cells stimulated with con A were cultivated with PGA1, and IFN-γ and TNF-α concentrations decreased upon PGA1 treatment. Taken together, these results suggest that the treatment with PGA1in vitro inhibits T-cell activation, especially Th1 cytokine production, in cattle.

Two novel Salp15-like immunosuppressant genes from salivary glands of Ixodes persulcatus Schulze tick.

Salp15, a 15-kDa tick salivary gland protein, is known for several suppressive activities against host immunity and critical functions for the transmission of Lyme borrelia in Ixodes scapularis and Ixodes ricinus, the major vectors found in North America and Western Europe. Salp15 inhibits the activation of cluster of differentiation (CD)4(+)T-cells through the repression of T-cell receptor (TCR)-triggered calcium fluxes and interleukin (IL)-2 production. Furthermore, Salp15 adheres to the spirochaeta and specifically interacts with its outer surface protein C. The binding of Salp15 to Borrelia burgdorferi protects it from antibody-mediated killing in vitro. The aim of this study is to identify the Salp15 genes in Ixodes persulcatus Schulze, the specific vector for human Lyme borreliosis in Japan. Two cDNA clones encoding the Salp15-like sequence were obtained from salivary glands of fed female ticks. These genes encode 135- and 132-amino acid proteins, designated Salp15 Iper-1 and Salp15 Iper-2, respectively, both having signal peptide sequences and predicted to be secretory proteins. Salp15 Iper-1 and -2 showed 51.8 and 68.2% similarity to I. scapularis Salp15, respectively. Reverse transcriptase PCR analysis showed that Salp15 Iper genes were expressed specifically in the salivary glands throughout life cycle stages of the ticks and were up-regulated by blood feeding. In the I. persulcatus-derived sequences, the C-terminal part, which is the binding domain to the CD4 molecule of T-cells in I. scapularis Salp15, was well conserved. In the future, it will be necessary to analyse immunosuppressive functions of I. persulcatus Salp15 and their interaction with Borrelia spp. in Japan.

Identification and characterization of antimicrobial peptide, defensin, in the taiga tick, Ixodes persulcatus.

Ixodes persulcatus is the primary vector for human tick-borne diseases in Japan. A cDNA library was constructed from whole body homogenates of fed nymphs of I. persulcatus. From this library, one cDNA encoding defensin-like antimicrobial peptide was identified. The amino-acid sequence showed high similarity to those of the defensins of other ticks and arthropods. I. persulcatus defensin mRNA transcripts were detected at all life cycle stages of fed ticks and found to be predominantly expressed in the midguts of adult female ticks, but not in the salivary glands, a finding corroborated by Western blotting analysis. To investigate the function of I. persulcatus defensin, we examined its antibacterial activity by evaluation of growth of several bacterial strains in the presence of the synthetic peptide. The defensin from I. persulcatus markedly inhibited the growth of Gram-positive bacteria including Staphylococcus aureus, Bacillus subtilis and Corynebacterium renale, but not Gram-negative bacteria except Escherichia coli O157. In conclusion, these results suggest that I. persulcatus defensin may be playing a significant role in the defence against microbes from bloodmeals.

Detection of the virulent Marek's disease virus genome from feather tips of wild geese in Japan and the Far East region of Russia.

Marek's disease (MD) virus (MDV) is known to cause malignant lymphomas in chickens. In 2001, we first reported an MD case in a white-fronted goose (Anser albifrons) in Japan. Therefore, the prevalence of MDV in the wild geese was surveyed by nested PCR using feather-tip samples in Japan and the Far East region of Russia, breeding habitats of geese migrating to Japan. MDV was detected in about 30% of analyzed white-fronted geese. Furthermore, by nucleotide sequence analysis, we confirmed that this MDV shows high homology to very virulent MDV, suggesting that highly virulent MDV is widespread in white-fronted geese migrating between Japan and Far East region of Russia.

Tumor necrosis factor-alpha up-regulation in spontaneously proliferating cells derived from bovine leukemia virus-infected cattle.

We previously reported that tumor necrosis factor alpha (TNF-alpha) was one of the cytokines that contributed to the leukemogenesis caused by bovine leukemia virus (BLV). To determine if the spontaneous cell proliferation observed in the late disease stages, such as persistent lymphocytosis and lymphosarcoma, correlated with the expression level of TNF-alpha, we analyzed the mRNA expression levels for TNF-alpha in spontaneously proliferating PBMCs derived from BLV-infected cattle. The mean mRNA expression level for TNF-alpha was higher in the spontaneously proliferating PBMCs derived from BLV-infected cattle than in non-spontaneously proliferating PBMCs from normal cattle. The TNF-alpha protein level in the PBMCs was determined by flow cytometric analysis, and it was noted that most of the cells expressing membrane-bound TNF-alpha in the spontaneously proliferating cells were CD5+ or sIgM+-cells. Additionally, in order to determine if this spontaneous proliferation can be blocked by anti-bovine TNF-alpha MAb, the spontaneously proliferating PBMCs from a BLV-infected cattle were cultured in the presence of the MAb. The addition of this MAb at the beginning of the 72 h-cultivation clearly inhibited spontaneous proliferation of cells in a dose-dependent manner, indicating the direct involvement of TNF-alpha in the spontaneous proliferation of PBMCs during the late disease stage. These data suggest that an aberrant expression of TNF-alpha might contribute to the progression of bovine leukosis in animals which develop persistent lymphocytosis of B-cells or B-cell lymphosarcoma.

Rapid detection and differentiation of Newcastle disease virus by real-time PCR with melting-curve analysis.

In order to rapidly detect and differentiate Newcastle disease virus (NDV) isolates, a method based on real-time PCR SYBR Green I melting-curve analysis of the fusion (F) protein gene was developed. The detection limit of real-time PCR was 9 x 10(2) plasmid copies and was 100 times more sensitive than conventional PCR. Thirty eight reference NDV strains were rapidly identified by their distinctive melting temperatures (T(m)s): 89.23 +/- 0.27 degrees C for velogenic strains, 90.17 +/- 0.35 degrees C for pigeon mesogenic strains, 91.25 +/- 0.14 degrees C for two lentogenic strains (B1 and Ishii). No amplification was detected from unrelated RNA samples by this method. This real-time PCR directly detected NDV from infected tissues and eliminated the gel electrophoretic step for analyzing PCR product using ethidium bromide. The total time for a PCR run was less than 1 hour. The results obtained in this study showed that the real-time PCR presented here is a good screening test for the identification of NDV.

Technical note: DNA typing for ovine MHC DRB1 using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).

The ovine major histocompatibilty complex (Ovar) class II DRB1 second exon was amplified by polymerase chain reaction (PCR) from DNA samples of 52 Suffolk sheep. Polymerase chain reaction products were characterized by the restriction fragment length polymorphism (RFLP) technique using nine restriction enzymes, RsaI, HaeIII, SacI, SacII, DdeI, NciI, Hin1I, EcoRI, and BstNI, yielding 13 types. Sequencing of cloned PCR products identified 16 Ovar-DRB1 alleles. Collectively, all PCR-RFLP patterns exactly matched those predicted from DNA sequences. These findings strongly indicate that the PCR-RFLP method using a combination of nine restriction endonucleases is a very powerful tool in Ovar typing.

Sequences and diversity of 17 new Ovar-DRB1 alleles from three breeds of sheep.

To investigate the genetic diversity of the sheep MHC (Ovar) class II DRB1 locus, we amplified exon 2 of Ovar-DRB1 alleles by polymerase chain reaction (PCR) and determined the nucleotide sequences of both resultant strands after cloning. In our study of a total of 97 sheep of three breeds, namely, Suffolk, Cheviot and Corriedale, we identified 18 previously published alleles and 17 new alleles. These alleles were 83.4 to 94.1% identical at the nucleotide level and 71.4 to 90.9% identical at the amino acid level to Ovar-DRB1*0101. We identified six new alleles in Cheviot sheep and 11 new alleles in Suffolk sheep. Furthermore, we identified 15, 6 and 1 allele in Suffolk, Cheviot and Corriedale sheep, respectively, that have only been found in these breeds to date. Analysis of the frequencies of the various Ovar-DRB1 alleles in each breed indicated that Ovar-DRB1*0702 was the most frequent allele in Suffolk sheep (23.9%), Ovar-DRB1*0203 was the most frequent allele in Cheviot sheep (27.5%) and Ovar-DRB1*0201 was the most frequent allele in Corriedale sheep (25.0%). A comparative analysis of the positions of polymorphic residues in the first extracellular domain of the DRB genes of sheep, humans and mice revealed an extraordinary similarity amongst the positions of polymorphic residues that are associated with the antigen recognition site (ARS). Moreover, the extent of polymorphism seems to be similar in sheep, humans and mice.