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CURATOR

PhD student, Universiti Kebangsaan Malaysia (UKM)

PINBOARD SUMMARY

To elucidate the molecular mechanisms of drug resistance and precocious development in E. tenella

The previous study of this parasite investigated the genetic basis and precocious development which resulted in the construction of a genetic linkage map from a cross between an arprinocid resistant line of the Eimeria tenella Weybridge strain and an attenuated E. tenella Wisconsin strain line. Additionally, various other genomic resources for E. tenella are currently available including the chromosome 1 and the whole genome sequences. These genome sequence data are publicly available and thus, it is currently feasible to employ the large-scale sequencing to identify the genetic factors (genes, promoters, etc.) that are associated with drug resistance and precocious development.

The mapping of the clones of E. tenella sequence reads to the available genome sequences will enable the identification of parent-specific SNPs in the genome of each clone. Sections of the genomes that demonstrate uni-parental genotypes can then be determined, and further analysis of these SNPs data will enable the identification of the genetic factors associated with drug resistance of precocious development.

The further characterisation of genetic factors will provide information regarding their potential functions and roles, and the molecular basis of drug resistance and precocious development to develop more effective controls for avian coccidiosis and other parasitic diseases.

8 ITEMS PINNED

Expression of host defense peptides in the intestine of Eimeria-challenged chickens.

Abstract: Avian coccidiosis is caused by the intracellular protozoan Eimeria, which produces intestinal lesions leading to weight gain depression. Current control methods include vaccination and anticoccidial drugs. An alternative approach involves modulating the immune system. The objective of this study was to profile the expression of host defense peptides such as avian beta-defensins (AvBDs) and liver expressed antimicrobial peptide 2 (LEAP2), which are part of the innate immune system. The mRNA expression of AvBD family members 1, 6, 8, 10, 11, 12, and 13 and LEAP2 was examined in chickens challenged with either E. acervulina, E. maxima, or E. tenella. The duodenum, jejunum, ileum, and ceca were collected 7 d post challenge. In study 1, E. acervulina challenge resulted in down-regulation of AvBD1, AvBD6, AvBD10, AvBD11, AvBD12, and AvBD13 in the duodenum. E. maxima challenge caused down-regulation of AvBD6, AvBD10, and AvBD11 in the duodenum, down-regulation of AvBD10 in the jejunum, but up-regulation of AvBD8 and AvBD13 in the ceca. E. tenella challenge showed no change in AvBD expression in any tissue. In study 2, which involved challenge with only E. maxima, there was down-regulation of AvBD1 in the ileum, AvBD11 in the jejunum and ileum, and LEAP2 in all 3 segments of the small intestine. The expression of LEAP2 was further examined by in situ hybridization in the jejunum of chickens from study 2. LEAP2 mRNA was expressed similarly in the enterocytes lining the villi, but not in the crypts of control and Eimeria challenged chickens. The lengths of the villi in the Eimeria challenged chickens were less than those in the control chickens, which may in part account for the observed down-regulation of LEAP2 mRNA quantified by PCR. Overall, the AvBD response to Eimeria challenge was not consistent; whereas LEAP2 was consistently down-regulated, which suggests that LEAP2 plays an important role in modulating an Eimeria infection.

Pub.: 19 May '17, Pinned: 30 Jul '17

A Novel Synthesis of the Efficient Anti-Coccidial Drug Halofuginone Hydrobromide.

Abstract: Background: Halofuginone hydrobromide (1) is recognized as an effective drug against several species of Eimeria (E.) in poultry. In this paper, we describe a convenient and low cost preparation method for the compound, as well as primary validation of its activity. Methods: First, 7-bromo-6-chloroquinazolin-4(3H)-one (2) was prepared from m-chlorotoluene by a conventional process, and then chloroacetone was creatively introduced in two steps. Finally, halofuginone hydrobromide (1) was obtained from 7-bromo-6-chloro-3-(3-cholroacetonyl) quinazolin-4(3H)-one (4) by a four-step reaction sequence including condensation, cyclization, deprotection and isomerization. The structures of the relative intermediates and target compound were characterized by melting point, IR, MS and ¹H-NMR. Besides, the protective effect of compound 1-supplemented chicken diet at doses of 6, 3 and 1.5 mg per 1 kg were evaluated on chickens infected with E. tenella, by reduction in mortality, weight loss, fecal oocyst excretion and gut pathology, respectively. Results: Halofuginone hydrobromide (1) was prepared successfully by and improved and innovative method based on traditional research. Moreover, the synthesized halofuginone hydrobromide significantly exhibited an anti-coccidial property. Conclusions: The fruitful work described in this Communication has resulted in halofuginone hydrobromide, which has a good pharmaceutical development prospects, becoming more available for large-scale production.

Pub.: 01 Jul '17, Pinned: 30 Jul '17

Eimeria tenella protein trafficking: differential regulation of secretion versus surface tethering during the life cycle.

Abstract: Eimeria spp. are intracellular parasites that have a major impact on poultry. Effective live vaccines are available and the development of reverse genetic technologies has raised the prospect of using Eimeria spp. as recombinant vectors to express additional immunoprotective antigens. To study the ability of Eimeria to secrete foreign antigens or display them on the surface of the sporozoite, transiently transfected populations of E. tenella expressing the fluorescent protein mCherry, linked to endogenous signal peptide (SP) and glycophosphatidylinositol-anchor (GPI) sequences, were examined. The SP from microneme protein EtMIC2 (SP2) allowed efficient trafficking of mCherry to cytoplasmic vesicles and following the C-terminal addition of a GPI-anchor (from surface antigen EtSAG1) mCherry was expressed on the sporozoite surface. In stable transgenic populations, mCherry fused to SP2 was secreted into the sporocyst cavity of the oocysts and after excystation, secretion was detected in culture supernatants but not into the parasitophorous vacuole after invasion. When the GPI was incorporated, mCherry was observed on the sporozites surface and in the supernatant of invading sporozoites. The proven secretion and surface exposure of mCherry suggests that antigen fusions with SP2 and GPI of EtSAG1 may be promising candidates to examine induction of protective immunity against heterologous pathogens.

Pub.: 06 Jul '17, Pinned: 30 Jul '17

Characterization of the antibody response in birds following infection with wild-type and attenuated strains of Eimeria tenella and Eimeria necatrix.

Abstract: Live vaccines containing attenuated parasite strains are increasingly used to control chicken coccidiosis. In this paper antibody responses elicited by infections with wild-type and attenuated strains of Eimeria tenella and Eimeria necatrix were characterized by immunoblotting and ELISA with homologous and heterologous antisera. Few differences between antisera from birds infected with wild and attenuated strains of E. tenella were evident in immunoblots conducted with merozoite antigen preparations from both E. tenella strains, however the reactivity of sera raised in birds infected with the wild-type strain was noticeably more intense. In ELISAs conducted with merozoite antigen preparations, antisera from birds infected with the wild-type strains of E. tenella and E. necatrix consistently produced a significantly higher (P<0.05) antibody response than antisera from birds infected with the attenuated strains. Likewise, avidity ELISAs conducted with the E. tenella strains demonstrated that antibodies in birds infected with the wild-type strain were of significantly higher avidity (P<0.05) than antibodies in birds infected with the attenuated strain. The differences in the antibody responses are probably due to changes in the attenuated strain as a result of selection for precocious development and the less severe tissue damage and inflammation of the intestine resulting from infection with the attenuated strain.

Pub.: 03 Nov '10, Pinned: 30 Jul '17