Flavonoid and Galloyl Glycosides Isolated from Saxifraga spinulosa and Their Antioxidative and Inhibitory Activities against Species That Cause Piroplasmosis.

Eight new flavonoid-based 3'-O-ß-d-glucopyranosides (1-8) and three new galloyl glucosides (9, 11, 12), were isolated from the aerial parts of Saxifraga spinulosa, along with 25 known compounds. The structures of the new compounds were elucidated by spectroscopic methods. Most of the isolated compounds exhibited potent DPPH radical-scavenging activities. Further, their inhibitory activities were evaluated against Babesia bovis, Babesia bigemina, Babesia caballi, and Theileria equi, protozoan parasites that cause piroplasmosis in livestock. The results indicated that several of these compounds showed growth-inhibitory effects on such organisms that cause piroplasmosis.

Comparative proteomic and bioinformatic analysis of Theileria luwenshuni and Theileria uilenbergi.

Theileria is an obligatory intraerythrocytic protozoan parasite that causes economic losses to the cattle, sheep and goats industry. However, very little information is available on the genomes, transcriptomes, and proteomes of the ovine parasites, Theileria luwenshuni and Theileria uilenbergi. Differences in protein expression between these species were investigated to better understand their biology. Parasites were digested with trypsin, and the resulting peptides labeled with isobaric tags for relative and absolute quantification, followed by LC-MS/MS. More than 670 proteins, classified into categories primarily related to cellular process (29.78%), metabolic process (28.80%), localization (5.22%) and biological regulation (5.00%), were identified. Seventy-one proteins were differentially expressed; T. luwenshuni had 39 proteins more highly expressed than in T. uilenbergi, whereas T. uilenbergi had 32 that were more highly expressed. Several proteins related to parasite virulence and invasion (cysteine proteinase, histone deacetylase, pyruvate kinase, small nuclear ribonucleoprotein and orotate phosphoribosyltransferase) were differentially expressed. Real-time quantitative PCR validated protein expression changes at the transcript level. This is the first report on protein expression for the two most economically important Theileria species in China, and our findings may provide novel opportunities for ovine and caprine theileriosis control.

Polymorphism analysis of Chinese Theileria sergenti using allele-specific polymerase chain reaction of the major piroplasm surface protein gene.

Theileria sergenti is a tick-borne parasite found in many parts of the world. The major piroplasm surface protein (MPSP), a conserved protein in all Theileria species, has been used as a marker for epidemiological and phylogenetic studies of benign Theileria species. In this study, Chinese species of T. sergenti were characterized by allele-specific polymerase chain reaction (PCR) and DNA sequence analysis of the MPSP gene. Using universal or allele-specific primer sets for PCR amplification of the MPSP gene, 98 of 288 cattle blood samples, collected from 6 provinces in China, were found to be positive. Among the positive samples, only 3 allelic MPSP gene types (Chitose [C]-, Ikeda [I]-, and buffeli [B]-type) were successfully amplified. Moreover, the results revealed that the majority of the parasites sampled in this study were C- and I-type (prevalence of 84 and 69%, respectively), whereas the B-type was less common (prevalence of 36%). Co-infections with C-, I-, and B-type T. sergenti also were found. An additional known allele, Thai-type, was not detected. Phylogenetic analysis based on the MPSP gene sequences, including 3 standard stocks generated in the laboratory ( T. sergenti Wenchuan, T. sergenti Ningxian, and T. sergenti Liaoyang), revealed that the isolates of Chinese sergenti were comprised of at least 4 allelic MPSP gene types, i.e., C-, I-, B1-, and B2-type, and these parasites with 6 MPSP types 1-5 and 7 were present in China.

Identification of Theileria uilenbergi immunodominant protein for development of an indirect ELISA for diagnosis of ovine theileriosis.

Theileriosis of small ruminants in the northwest of China is a protozoan disease that restricts the development of the livestock industry. The disease is caused by infection with Theileria uilenbergi and Theilerialuwenshuni, both of which are transmitted by ixodid Heamaphysalis ticks. The development of serological tools as a means of integrated control of the disease is an urgent and important requirement. Here we describe the identification and partial recombinant expression of a T.uilenbergi immunodominant protein (TuIP), which was identified by immunoscreening of a merozoite cDNA library. Using the recombinant TuIP (rTuIP), a novel indirect ELISA was established using 329 negative serum samples to determine the cut-off value. The internal quality control revealed satisfactory stability and repeatability of the assay. Preliminary validation using 128 positive and 48 negative reference samples demonstrated that the rTuIP ELISA is able to detect T. uilenbergi infection with high sensitivity and specificity. No cross-reactivity was found in sera from animals infected with Theileria lestoquardi, Babesia sp. China or Anaplasma ovis. Furthermore, circulating antibodies were detected in sera collected from endemic regions in China. Analyses of the antibody responses of experimentally infected animals demonstrated that tick infestation resulted in a sharply rising and stronger production of specific antibodies against TuIP while inoculation with infected blood induced an earlier production of TuIP-specific antibodies. The persistence of the TuIP-specific antibodies lasted more than 100days p.i. These data indicate the usefulness of the TuIP antigen for the development of diagnostic methods and as a potential candidate for vaccine design.

Detección molecular de patógenos emergentes de importancia médica y veterinaria en garrapatas capturadas sobre caballos domésticos / Molecular detection of emerging pathogens of medical and veterinary importance in ticks found in domestic horses

En la actualidad son varias las especies de patógenos emergentes de importancia médica y veterinaria transmitidos por garrapatas. Los estudios sobre estos agentes y sus enfermedades han sido escasos en Cuba. Conocer la presencia de algunos de estos patógenos en garrapatas cubanas que afectan el ganado equino. Se procesaron 95 garrapatas colectadas de caballos domésticos, conservadas en alcohol e identificadas taxonómicamente según claves convencionales. A cada una se le realizó extracción de ADN y posteriormente diferentes reacciones en cadena de la polimerasa utilizando cebadores específicos para los grupos microbianos Borrelia burgdorferi sensu lato, Anaplasma-Ehrlichia, y Babesia-Theileria. Cada uno de los productos de las reacciones en cadena de la polimerasa fue sometido a hibridaciones en línea reversa utilizando sondas para cada grupo en cuestión, así como específicas para las principales especies de estos. Las garrapatas estudiadas pertenecían a las especies Dermacentor (Anocentor) nitens (60 por ciento), Amblyomma cajennense (38 por ciento) y Rhipicephalus (Boophilus) microplus (2 por ciento). Se detectaron 7 garrapatas Dermacentor (Anocentor) nitens infectadas con bacterias del grupo Anaplasma/Ehrlichia, y no se pudo identificar la especie en cuestión con las sondas utilizadas. Una de estas garrapatas estaba además coinfectada con Babesia bovis. Se sugiere la circulación de una nueva especie de Anaplasma o Ehrlichia no reportada antes en Cuba, por lo que se necesita estudiar un número mayor de garrapatas, así como la incorporación de nuevas sondas en la hibridación en línea reversa u otras metodologías que permitan conocer con exactitud las especies que pudiesen afectar hoy día los caballos domésticos.

At present, there are several tick-borne emerging pathogen species of medical and veterinary importance. Few studies on these agents and its diseases have been made in Cuba. To determine the presence of some of these pathogens in Cuban ticks existing in the equine cattle. Ninety five ticks collected from domestic use horses were processed, preserved in alcohol and taxonomically identified according to the set classifications. Their DNA was extracted and subjected to several polymerase chain reactions with specific primers for microbial groups Borrelia burgdorferi sensu lato, Anaplasma-Ehrlichia, y Babesia-Theileria. Each of the products from polymerase chain reactions underwent reverse line blot hybridation using probes for each group as well as specific probes for the main species included in these groups. The studied ticks belonged to Dermacentor (Anocentor) nitens (60 percent), Amblyomma cajennense (38 percent) y Rhipicephalus (Boophilus) microplus (2 percent). Seven Dermacentor (Anocentor) nitens ticks infested with Anaplasma/Ehrlichia bacteria were detected but the species in question could not be detected by the used probes. One of these ticks was also co-infested with Babesia bovis. It is suggested that a new species of Anaplasma o Ehrlichia, not reported in Cuba before now, is circulating, so studying a higher number of ticks is needed and new probes in reverse line blot hybridation or other methodologies must be incorporated to allow exactly determining the species that may affect the Cuban domestic horses at present.

Detección molecular de patógenos emergentes de importancia médica y veterinaria en garrapatas capturadas sobre caballos domésticos / Molecular detection of emerging pathogens of medical and veterinary importance in ticks found in domestic horses

En la actualidad son varias las especies de patógenos emergentes de importancia médica y veterinaria transmitidos por garrapatas. Los estudios sobre estos agentes y sus enfermedades han sido escasos en Cuba. Conocer la presencia de algunos de estos patógenos en garrapatas cubanas que afectan el ganado equino. Se procesaron 95 garrapatas colectadas de caballos domésticos, conservadas en alcohol e identificadas taxonómicamente según claves convencionales. A cada una se le realizó extracción de ADN y posteriormente diferentes reacciones en cadena de la polimerasa utilizando cebadores específicos para los grupos microbianos Borrelia burgdorferi sensu lato, Anaplasma-Ehrlichia, y Babesia-Theileria. Cada uno de los productos de las reacciones en cadena de la polimerasa fue sometido a hibridaciones en línea reversa utilizando sondas para cada grupo en cuestión, así como específicas para las principales especies de estos. Las garrapatas estudiadas pertenecían a las especies Dermacentor (Anocentor) nitens (60 por ciento), Amblyomma cajennense (38 por ciento) y Rhipicephalus (Boophilus) microplus (2 por ciento). Se detectaron 7 garrapatas Dermacentor (Anocentor) nitens infectadas con bacterias del grupo Anaplasma/Ehrlichia, y no se pudo identificar la especie en cuestión con las sondas utilizadas. Una de estas garrapatas estaba además coinfectada con Babesia bovis. Se sugiere la circulación de una nueva especie de Anaplasma o Ehrlichia no reportada antes en Cuba, por lo que se necesita estudiar un número mayor de garrapatas, así como la incorporación de nuevas sondas en la hibridación en línea reversa u otras metodologías que permitan conocer con exactitud las especies que pudiesen afectar hoy día los caballos domésticos(AU)

At present, there are several tick-borne emerging pathogen species of medical and veterinary importance. Few studies on these agents and its diseases have been made in Cuba. To determine the presence of some of these pathogens in Cuban ticks existing in the equine cattle. Ninety five ticks collected from domestic use horses were processed, preserved in alcohol and taxonomically identified according to the set classifications. Their DNA was extracted and subjected to several polymerase chain reactions with specific primers for microbial groups Borrelia burgdorferi sensu lato, Anaplasma-Ehrlichia, y Babesia-Theileria. Each of the products from polymerase chain reactions underwent reverse line blot hybridation using probes for each group as well as specific probes for the main species included in these groups. The studied ticks belonged to Dermacentor (Anocentor) nitens (60 percent), Amblyomma cajennense (38 percent) y Rhipicephalus (Boophilus) microplus (2 percent). Seven Dermacentor (Anocentor) nitens ticks infested with Anaplasma/Ehrlichia bacteria were detected but the species in question could not be detected by the used probes. One of these ticks was also co-infested with Babesia bovis. It is suggested that a new species of Anaplasma o Ehrlichia, not reported in Cuba before now, is circulating, so studying a higher number of ticks is needed and new probes in reverse line blot hybridation or other methodologies must be incorporated to allow exactly determining the species that may affect the Cuban domestic horses at present(AU)

The beta-tubulin gene of Babesia and Theileria parasites is an informative marker for species discrimination.

A fragment of the beta-tubulin gene was polymerase chain reaction (PCR) amplified from genomic DNAs of Babesia bovis, Babesia bigemina, Babesia divergens, Babesia major, Babesia caballi, Babesia equi, Babesia microti, Theileria annulata and Theileria sergenti. Single amplification products were obtained for each of these species, but the size of the amplicons varied from 310 to 460 bp. Sequence analysis revealed that this variation is due to the presence of a single intron, which ranged from 20 to 170 bp. The extensive genetic variability at the beta-tubulin locus has been exploited to develop two types of species identification assays. The first assay can be used on samples containing mostly parasite DNA, like those prepared from infected erythrocytes. Following PCR amplification, the species identification is obtained directly from the size of the products (for Babesia species infecting human or horse) or using a simple PCR-restriction fragment length polymorphism (RFLP) protocol (for Babesia species infecting cattle). The second assay can be used on samples prepared from whole blood, that contain both parasite and host DNAs. In this case, due to the strong conservation of the beta-tubulin gene, co-amplification of a gene fragment from the host DNA was observed. A nested PCR assay was developed for the specific amplification of parasite DNA, using a primer designed to span the exon-intron boundary. Direct identification of Babesia species infecting human and horse is again obtained after the electrophoretic separation of the amplification products, while for Babesia and Theileria species infecting cattle, differentiation is based on a nested PCR-RFLP protocol. These methods may be used for the simultaneous identification of horses and cattle carrying multiple parasites by means of a single PCR or using the PCR-RFLP protocol.

Theileriosis in a Missouri beef herd caused by Theileria buffeli: case report, herd investigation, ultrastructure, phylogenetic analysis, and experimental transmission.

A 6-year-old Simmental cow infected with Theileria buffeli had a clinical disease characterized by theilerial parasitemia, macrocytic normochromic anemia with acanthocytosis and spherocytosis, lymphoid hyperplasia (lymphocytosis, edematous lymphadenomegaly), dysproteinemia, evidence of liver disease, and a low serum antibody titer against T. buffeli. The cow was in a herd in which all cattle originated in Missouri; 22/75 (29%) of cattle had a theilerial parasitemia and 26/75 (35%) had titers to T. buffeli of > or =1:160. Classification of the Missouri bovine organism as T. buffeli was based on DNA sequencing and comparison to sequences for T. buffeli and Theileria sp. type A obtained from GenBank. Intraerythrocytic veils and piroplasms were seen during transmission electron microscopy. The organism was successfully transmitted to two splenectomized calves, which developed mild anemias while parasitemic. Blood from the second calf was used as the source of T. buffeli antigen for an indirect immunofluorescence antibody test. Theilerial isolates from a Missouri white-tailed deer were also sequenced and resembled Theileria sp. types F and G and were not consistent with the bovine organism.

Genetic diversity of major piroplasm surface protein genes and their allelic variants of Theileria parasites in Thai cattle.

Twenty-eight field isolated Theileria parasite DNAs obtained from dairy and beef cattle in distinct geographical areas of Thailand were characterized by using polymerase chain reaction (PCR) amplification with six sets of oligonucleotide primers. Three sets of them were modified from two genes of immunodominant major piroplasm surface protein (MPSP) coding for 32 kDa (p32) of T. sergenti and 33/34 kDa (p33/34) of T. buffeli, and MPSP of Theileria spp.(Thai-isolate). The other three sets of primers were basically generated from three alleles of MPSP which were specific for Japanese T. sergenti-Ikeda stock (I-type), Japanese T. sergenti-Chitose stock (C-type) and Australian T. buffeli-Warwick stock (B1-type), respectively. The results indicated that 14 out of 28 isolates were amplified by the Thai-specific primer whereas 6 isolates were amplified by the p32 specific primer and the other 5 isolates were amplified by the p32 and Thai-specific primers. In addition, by using the allele-specific PCR, 14 out of 28 isolates contained C-type MPSP whereas 3 isolates contained B1 type parasites. Interestingly, 20 out of 28 isolates could be amplified by the Thai-specific primer. The majority of Theileria parasites distributed in Thailand contained Thai type parasites, whereas C-type parasites showed the mixed population with B1 and Thai type parasites. No I type parasite was detected.

Molecular cloning and characterisation of 23-kDa piroplasm surface proteins of Theileria sergenti and Theileria buffeli.

The cDNA encoding a 23-kDa piroplasm membrane protein (p23) of Theileria sergenti Chitose (C)-type was isolated and its nucleotide sequence was determined. The gene encodes a polypeptide of 223 aa with a 28 residue N-terminal signal sequence and a hydrophobic, valine-rich, C-terminal transmembrane domain, as deduced from its nucleotide sequence. Southern blot hybridisation analysis proved that p23 gene was a single copy gene and had allelic forms of the gene in the parasite population. By PCR, the open reading frames of T. sergenti Ikeda (I)-type and Theileria buffeli (B)-type p23 were amplified from genomic DNA and their nucleotide sequences were also determined. Comparison of C-type sequence with that of I-type and B-type revealed 90.5% and 93.5% sequence similarity, respectively, at the aa level. These results suggest that a conserved molecule in these benign Theileria spp. could be a candidate antigen for the development of an anti-piroplasm vaccine.

Phylogenetic relationships of the benign Theileria species in cattle and Asian buffalo based on the major piroplasm surface protein (p33/34) gene sequences.

In order to examine the taxonomic relationship of Theileria sp. of Asian buffalo to the benign Theileria spp. of cattle, we sequenced and compared the major piroplasm protein (p33/34) genes of these parasites. The two consensus sequences determined for the buffalo parasite were of the same length (852 bp) and showed >80% identity with the sequences of the homologous genes (849 bp) in the cattle parasites. Alignment of the inferred aa sequences with those of Theileria sergenti and Theileria buffeli predicted that there is an insertion of a single residue at the N-terminus in the inferred polypeptide of the buffalo parasite. Phylogenetic analyses based on the aa sequences suggested that Theileria sp. of the Asian buffalo should be classified within the benign Theileria parasite group as a separate species from the cattle parasites. Based on this, we propose a rearrangement of the currently used classification for the benign Theileria species in cattle and Asian buffalo.

[Cloning and sequencing of p33 in a Korean isolate of Theileria sergenti].

The gene encoding the 33 kDa piroplasm surface protein of Theileria sergenti isolated in Korea was cloned and the nucleotide sequence was determined by dideoxy chain termination method. The cloned gene corresponds to 869 bp encoding an open reading frame 283 amino acids. Comparison of the sequence between Korean and Japanese isolates showed 99.4% homology rate in the nucleotide sequence and 98.9% homology rate in the amino acid sequence.

Isolation and characterization of gangliosides from Theileria sergenti.

The gangliosides of Theileria sergenti piroplasms were isolated and analyzed by thin-layer chromatography (TLC) and TLC immunostaining test. Four species of gangliosides, designated as G-1, G-2, G-3, and G-4, were separated on TLC. G-1, G-2, G-3, and G-4 ganglioside showed the same mobility as GM3, sialosylparagloboside (SPG), i-active ganglioside, and I-active ganglioside on the TLC plate, respectively. In order to characterize the molecular species of gangliosides from T. sergenti, G-1, G-2, G-3, and G-4 gangliosides were purified and tested by TLC immunostaining test with monoclonal antibodies against gangliosides. G-1 ganglioside had reactivity to anti-GM3 monoclonal antibody. G-2 gave reaction with monoclonal antibody to SPG containing N-glycolylneuraminic acid (NeuGc). G-3 showed reactivity to the anti-i-active ganglioside (NeuGc) monoclonal antibody. G-4 was recognized by the monoclonal antibody which reacts with I-active ganglioside (NeuGc). In addition, sialic acid moiety of the gangliosides from T. sergenti piroplasms was also analyzed. N-acetylneuraminic acid-containing gangliosides were hardly detectable in T. sergenti piroplasms. Gangliosides from T. sergenti (G-1, G-2, G-3, and G-4) carried only NeuGc as their sialic acid moiety. These results suggest that G-1, G-2, G-3, and G-4 gangliosides are GM3 (NeuGc) [NeuGc alpha 2-3Gal beta 1-4Glc beta 1-Cer], SPG (NeuGc) [NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer], i-active ganglioside (NeuGc) [NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer], and I-active ganglioside(NeuGc) [NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3 (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6) Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer], respectively.

Analysis of neutral glycosphingolipids of Theileria sergenti piroplasms.

Neutral glycosphingolipids (GSLs) were isolated from a lipid extract of Theileria sergenti piroplasms and analyzed by thin-layer chromatography (TLC) and liposome immune lysis assay (LILA); two predominant GSLs, designated as N-1 and N-2 were separated on TLC. N-1 GSL showed the same mobility as lactosylceramide (LacCer) on the TLC plate. On the other hand, the mobility of N-2 GSL on the TLC plate was identical to that of galactosylparagloboside. In order to characterize the molecular species of neutral GSLs from T. sergenti, N-1 and N-2 GSLs were tested by LILA with antibodies against LacCer and galactosylparagloboside, respectively, N-1 GSL had reactivity to anti-LacCer antibody and N-2 reacted with the antibody to galactosylparagloboside. These results suggest that N-1 and N-2 GSLs are LacCer (Gal beta 1-4Glc beta 1-1Cer) and galactosylparagloboside (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer), respectively.