Phenotypic and genetic variation in the response of chickens to Eimeria tenella induced coccidiosis.

BACKGROUND: Coccidiosis is a major contributor to losses in poultry production. With emerging constraints on the use of in-feed prophylactic anticoccidial drugs and the relatively high costs of effective vaccines, there are commercial incentives to breed chickens with greater resistance to this important production disease. To identify phenotypic biomarkers that are associated with the production impacts of coccidiosis, and to assess their covariance and heritability, 942 Cobb500 commercial broilers were subjected to a defined challenge with Eimeria tenella (Houghton). Three traits were measured: weight gain (WG) during the period of infection, caecal lesion score (CLS) post mortem, and the level of a serum biomarker of intestinal inflammation, i.e. circulating interleukin 10 (IL-10), measured at the height of the infection. RESULTS: Phenotypic analysis of the challenged chicken cohort revealed a significant positive correlation between CLS and IL-10, with significant negative correlations of both these traits with WG. Eigenanalysis of phenotypic covariances between measured traits revealed three distinct eigenvectors. Trait weightings of the first eigenvector, (EV1, eigenvalue = 59%), were biologically interpreted as representing a response of birds that were susceptible to infection, with low WG, high CLS and high IL-10. Similarly, the second eigenvector represented infection resilience/resistance (EV2, 22%; high WG, low CLS and high IL-10), and the third eigenvector tolerance (EV3, 19%; high WG, high CLS and low IL-10), respectively. Genome-wide association studies (GWAS) identified two SNPs that were associated with WG at the suggestive level. CONCLUSIONS: Eigenanalysis separated the phenotypic impact of a defined challenge with E. tenella on WG, caecal inflammation/pathology, and production of IL-10 into three major eigenvectors, indicating that the susceptibility-resistance axis is not a single continuous quantitative trait. The SNPs identified by the GWAS for body weight were located in close proximity to two genes that are involved in innate immunity (FAM96B and RRAD).

Heterobucephalopsine and prosorhynchine trematodes (Digenea: Bucephalidae) from teleost fishes of Moreton Bay, Queensland, Australia, with the description of two new species.

Eight species of the trematode family Bucephalidae Poche, 1907 are reported from teleost fishes in Moreton Bay, Queensland, Australia. Heterobucephalopsis yongi n. sp. is described from Gymnothorax eurostus (Muraenidae); the new form is distinguished from its congeners in the possession of a tiny cirrus-sac relative to body length, the length of the caecum, the position of the mouth and pharynx, and the position of the testes and ovary. Two known species of Dollfustrema Eckmann, 1934, D. durum Nolan, Curran, Miller, Cutmore, Cantacessi & Cribb, 2015 and D. gibsoni Nolan & Cribb, 2010, are reported from Gymnothorax pseudothyrsoideus (Bleeker) (Muraenidae); although both species were described from Australian waters, this represents the first reports from Moreton Bay and G. pseudothyrsoideus. Four species of Prosorhynchus Odhner, 1905 are reported, including one new, P. brayi n. sp., which is described from Epinephelus coioides (Hamilton) (Serranidae); P. brayi n. sp. is distinguished from its congeners in the possession of vitelline follicles in a confluent arc distinctly posterior to a conical rhynchus, uterine coils that do not extend anterior to the vitelline arc, contiguous testes, a cirrus-sac that reaches anteriorly to at least the level of the posterior testis and a short excretory vesicle. Three known species of Prosorhynchus are reported from Australia, for the first time: P. luzonicus Velasquez, 1959 and P. maternus Bray & Justine, 2006 from E. coioides and Prosorhynchus platycephali (Yamaguti, 1934) Srivastava, 1938 from Ambiserrula jugosa (McCulloch) and Inegocia japonica (Cuvier) (Platycephalidae). Skrjabiniella Issaitschikow, 1928 is re-recognised for new specimens of Skrjabiniella uniporus (Ozaki, 1924) n. comb. collected from Conger cinereus Rüppell (Congridae); three additional species of Prosorhynchus are considered members of this genus, two of which are synonymised with S. uniporus.

Molecular approaches to trematode systematics: 'best practice' and implications for future study.

To date, morphological analysis has been the cornerstone to trematode systematics. However, since the late-1980s we have seen an increased integration of genetic data to overcome problems encountered when morphological data are considered in isolation. Here, we provide advice regarding the 'best molecular practice' for trematode taxonomy and systematic studies, in an attempt to help unify the field and provide a solid foundation to underpin future work. Emphasis is placed on defining the study goals and recommendations are made regarding sample preservation, extraction methods, and the submission of molecular vouchers. We advocate generating sequence data from all parasite species/host species/geographic location combinations and stress the importance of selecting two independently evolving loci (one ribosomal and one mitochondrial marker). We recommend that loci should be chosen to provide genetic variation suitable to address the question at hand and for which sufficient 'useful' comparative sequence data already exist. Quality control of the molecular data via using proof-reading Taq polymerase, sequencing PCR amplicons using both forward and reverse primers, ensuring that a minimum of 85% overlap exists when constructing consensus sequences, and checking electropherograms by eye is stressed. We advise that all genetic results are best interpreted using a holistic biological approach, which considers morphology, host identity, collection locality, and ecology. Finally, we consider what advances next-generation sequencing holds for trematode taxonomy and systematics.

Impact of experimental hookworm infection on the human gut microbiota.

The interactions between gastrointestinal parasitic helminths and commensal bacteria are likely to play a pivotal role in the establishment of host-parasite cross-talk, ultimately shaping the development of the intestinal immune system. However, little information is available on the impact of infections by gastrointestinal helminths on the bacterial communities inhabiting the human gut. We used 16S rRNA gene amplification and pyrosequencing to characterize, for the first time to our knowledge, the differences in composition and relative abundance of fecal microbial communities in human subjects prior to and following experimental infection with the blood-feeding intestinal hookworm, Necator americanus. Our data show that, although hookworm infection leads to a minor increase in microbial species richness, no detectable effect is observed on community structure, diversity or relative abundance of individual bacterial species.

First molecular characterization of Giardia duodenalis from goats in Malaysia.

In the present study, 310 faecal samples from goats from eight different farms in Malaysia were tested for the presence of Giardia using a PCR-coupled approach. The nested PCR for SSU amplified products of the expected size (∼200 bp) from 21 of 310 (6.8%) samples. Sixteen of these 21 products could be sequenced successfully and represented six distinct sequence types. Phylogenetic analysis of the SSU sequence data using Bayesian Inference (BI) identified Giardia assemblages A, B and E. The identification of the 'zoonotic' assemblages A and B suggests that Giardia-infected goats represent a possible reservoir for human giardiasis in Malaysia.

Mutation scanning-based analysis of anisakid larvae from Sillago flindersi from Bass Strait, Australia.

Anisakidosis is an important fish-borne disease caused by the larvae of anisakid nematodes, which affects humans and a range of other animals. The accurate identification of members of this nematode group is central to investigating the epidemiology of the parasites and in the surveillance and control of anisakidosis. It is now well known that morphological identification alone does not allow specific identification, particularly of larval stages. To better understand the epidemiology of anisakid nematodes in southern Australian fishes and the potential risks posed to human health, a survey of 50 specimens of the commercially important fish, Sillago flindersi, from Bass Strait, Australia was conducted. We characterised anisakid larvae by PCR-coupled mutation scanning, sequencing and phylogenetic analyses of the first and second internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA. This study revealed that 92% of the S. flindersi examined were infected with anisakids (n=194), which were represented by seven genotypes. Phylogenetic analyses of the genotypes defined herein, together with reference sequence for Anisakis pegreffii and Hysterothylacium sp. from public databases (i.e. GenBank), revealed the presence of A. pegreffii (n=24), Hysterothylacium larval type IV (n=90) and Hysterothylacium larval type VIII (n=80) in S. flindersi. Thus, the PCR-coupled mutation scanning approach employed herein is an effective tool for the genetic characterisation of anisakid nematodes for diagnostic and analytical purposes (nucleotide sequences reported in this paper are available in the GenBank database under accession nos. JN631796-809).

Detection of diarrhoeal pathogens in human faeces using an automated, robotic platform.

Infectious diarrhoeal diseases represent a major socio-economic burden to humans, and are linked to a range of pathogens, including viruses, bacteria and protists. The accurate detection of such pathogens is central to control. However, detection often relies on methods that have limited diagnostic sensitivity and specificity. Here, we assessed an automated, robotic platform for the simultaneous detection of eight major pathogens associated with infectious diarrhoea. Genomic DNA samples (n = 167) from faeces from humans with diarrhoea and diagnosed as cryptosporidiosis, and 100 uninfected control subjects, were tested for adenovirus 40/41, norovirus, Clostridium difficile, Campylobacter, Salmonella, Shigella, Cryptosporidium and Giardia by multiplexed-tandem PCR, and also characterized by single-strand conformation polymorphism analysis (SSCP) and selective sequencing. All 167 samples tested positive for Cryptosporidium, five for adenovirus 40/41, four for Campylobacter, three for C. difficile and seven for Shigella spp., with no false positive results for any assay. The automated PCR exhibited a high sensitivity, with <10 individual pathogens being readily detected. The robotic detection platform assessed here represents a sensitive, high-throughput tool for key pathogens linked to infectious diarrhoea in humans. This platform requires little molecular biological expertise and is well suited to various diagnostic facilities and settings.

A practical, bioinformatic workflow system for large data sets generated by next-generation sequencing.

Transcriptomics (at the level of single cells, tissues and/or whole organisms) underpins many fields of biomedical science, from understanding the basic cellular function in model organisms, to the elucidation of the biological events that govern the development and progression of human diseases, and the exploration of the mechanisms of survival, drug-resistance and virulence of pathogens. Next-generation sequencing (NGS) technologies are contributing to a massive expansion of transcriptomics in all fields and are reducing the cost, time and performance barriers presented by conventional approaches. However, bioinformatic tools for the analysis of the sequence data sets produced by these technologies can be daunting to researchers with limited or no expertise in bioinformatics. Here, we constructed a semi-automated, bioinformatic workflow system, and critically evaluated it for the analysis and annotation of large-scale sequence data sets generated by NGS. We demonstrated its utility for the exploration of differences in the transcriptomes among various stages and both sexes of an economically important parasitic worm (Oesophagostomum dentatum) as well as the prediction and prioritization of essential molecules (including GTPases, protein kinases and phosphatases) as novel drug target candidates. This workflow system provides a practical tool for the assembly, annotation and analysis of NGS data sets, also to researchers with a limited bioinformatic expertise. The custom-written Perl, Python and Unix shell computer scripts used can be readily modified or adapted to suit many different applications. This system is now utilized routinely for the analysis of data sets from pathogens of major socio-economic importance and can, in principle, be applied to transcriptomics data sets from any organism.

Barcoding of Giardia duodenalis isolates and derived lines from an established cryobank by a mutation scanning-based approach.

We barcoded 25 in vitro isolates (representing 92 samples) of Giardia duodenalis from humans and other animals, which have been assembled by the Upcroft team at the Queensland Institute of Medical Research over a period of almost three decades. We used mutation scanning-coupled sequencing of loci in the triosephosphate isomerase, glutamate dehydrogenase and ß-giardin genes, combined with phylogenetic analysis, to genetically characterise them. Specifically, the isolates (n514) of G. duodenalis from humans from Australia (AD113; BRIS/83/HEPU/106; BRIS/87/HEPU/713; BRIS/89/HEPU/1003; BRIS/92/HEPU/1541; BRIS/92/HEPU/1590; BRIS/92/HEPU/2443; BRIS/93/HEPU/1706), Malaysia (KL/92/IMR/1106) and Afghanistan (WB), a cat from Australia (BAC2), a sheep from Canada (OAS1) and a sulphur-crested cockatoo from Australia (BRIS/95/HEPU/2041) represented assemblage A (sub-assemblage AI-1, AI-2 or AII-2); isolates (n510) from humans from Australia (BRIS/91/HEPU/1279; BRIS/92/HEPU/2342; BRIS/92/HEPU/2348; BRIS/93/HEPU/1638; BRIS/93/HEPU/1653; BRIS/93/HEPU/1705; BRIS/93/HEPU/1718; BRIS/93/HEPU/1727), Papua New Guinea (BRIS/92/HEPU/1487) and Canada (H7) represented assemblage B (sub-assemblage BIV) and an isolate from cattle from Australia (BRIS/92/HEPU/1709) had a match to assemblage E. Isolate BRIS/90/HEPU/1229 from a human from Australia was shown to represent a mixed population of assemblages A and B. These barcoded isolates (including stocks and derived lines) now allow direct comparisons of experimental data among laboratories and represent a massive resource for transcriptomic, proteomic, metabolic and functional genomic studies using advanced molecular technologies.

A first insight into the genotypes of Echinococcus granulosus from humans in Mongolia.

Polymerase chain reaction (PCR)-based single-strand conformation polymorphism (SSCP) and targeted sequencing were employed to genetically classify Echinococcus granulosus cysts from humans from 12 provinces in Mongolia using two DNA loci, designated pcox-1 and pnad-1, within the mitochondrial cytochrome c oxidase subunit 1 (cox-1) and NADH dehydrogenase subunit 1 (nad-1) genes, respectively. SSCP analysis of pcox-1 and pnad-1 amplicons produced from genomic DNA samples from individual E. granulosus cysts (n = 50) from individual humans displayed four distinct electrophoretic profiles for each pcox-1 and pnad-1. The direct sequencing of selected amplicons representing each of these profiles defined four distinct sequence types for each locus, present in four different combinations (designated as haplotypes M1-M4) for all 50 cyst isolates. Phylogenetic analysis of concatenated sequence data for these four haplotypes, including well-defined reference sequences, inferred that 68% of the cyst isolates belonged to the G1-G3 complex of E. granulosus (or E. granulosus sensu stricto), whereas the remaining (32%) were linked to the G6-G10 complex (or Echinococcus canadensis). Humans infected with E. granulosus cysts of the G1-G3 complex originated mainly from the eastern regions of Mongolia, whereas those harbouring cysts of the G6-G10 complex were from the western part of this country. The present study provides a first glimpse of the genetic composition of E. granulosus from humans in Mongolia, and forms a foundation for future studies of the epidemiology and ecology of the parasite(s) in animals and humans in this and surrounding countries.

Genetic and biological characterisation of three cryptic Eimeria operational taxonomic units that infect chickens (Gallus gallus domesticus).

More than 68 billion chickens were produced globally in 2018, emphasising their major contribution to the production of protein for human consumption and the importance of their pathogens. Protozoan Eimeria spp. are the most economically significant parasites of chickens, incurring global costs of more than UK £10.4 billion per annum. Seven Eimeria spp. have long been recognised to infect chickens, with three additional cryptic operational taxonomic units (OTUs) first described more than 10 years ago. As the world's farmers attempt to reduce reliance on routine use of antimicrobials in livestock production, replacing drugs that target a wide range of microbes with precise species- and sometimes strain-specific vaccines, the breakthrough of cryptic genetic types can pose serious problems. Consideration of biological characteristics including oocyst morphology, pathology caused during infection and pre-patent periods, combined with gene-coding sequences predicted from draft genome sequence assemblies, suggest that all three of these cryptic Eimeria OTUs possess sufficient genetic and biological diversity to be considered as new and distinct species. The ability of these OTUs to compromise chicken bodyweight gain and escape immunity induced by current commercially available anticoccidial vaccines indicates that they could pose a notable threat to chicken health, welfare, and productivity. We suggest the names Eimeria lata n. sp., Eimeria nagambie n. sp. and Eimeria zaria n. sp. for OTUs x, y and z, respectively, reflecting their appearance (x) or the origins of the first isolates of these novel species (y, z).

Highly sensitive non-isotopic restriction endonuclease fingerprinting of nucleotide variability in the gp60 gene within Cryptosporidium species, genotypes and subgenotypes infective to humans, and its implications.

The high-resolution analysis of genetic variation has major implications for the identification of parasites and micro-organisms to species and subspecies as well as for population genetic and epidemiological studies. In this study, we critically assessed the effectiveness of a PCR-based restriction endonuclease fingerprinting (REF) method for the detection of mutations in the 60 kDa glycoprotein gene (gp60) of Cryptosporidium, a genus of parasitic protists of major human and animal health importance globally. This gene displays substantial intraspecific variability in sequence, particularly in a TCA (perfect and imperfect) microsatellite region, is present as a single copy in the nuclear genome and is used widely as a marker in molecular epidemiological studies of Cryptosporidium hominis and C. parvum, the two predominant species that infect humans. The results of this study demonstrated an exquisite capacity of REF to detect nucleotide variability in the gp60 gene within each of the two species. The differentiation of genotypes/subgenotypes based on REF analysis was supported by targeted sequencing, allowing the detection of levels of variation as low as a single-nucleotide transversion for amplicons of approximately 1 kb in size. The high-throughput potential and relatively low-cost of REF make it a particularly useful tool for large-scale genetic analyses of C. hominis and C. parvum. REF could also be utilized for comparative surveys of genetic variability across large nuclear genomic regions. Such analyses of Cryptosporidium in clinical and environmental samples by REF have important implications for identifying sources of infection, modes of transmission and/or possible infectivity to humans, thus assisting in the surveillance and control of cryptosporidiosis. Given its excellent mutation detection capacity, REF should find broad applicability to various single-copy genes as well as a wide range of other protozoan and metazoan parasites.

Analysis of nucleotide variation within the triose-phosphate isomerase gene of Giardia duodenalis from sheep and its zoonotic implications.

This study explored the genetic composition of Giardia in fecal samples from 284 individual lambs on pasture-based sheep farms in three regions of Victoria, Australia. An approach, combining targeted sequencing, phylogenetic analysis and PCR-coupled restriction endonuclease fingerprinting, was used to identify and genetically categorize Giardia present in 43 (15.1%) of the 284 samples and to infer their zoonotic potential. The specific identity and genetic classification were based on the phylogenetic analysis of sequence data for a portion of the triose-phosphate isomerase gene. Fourteen different sequence variants (including seven sequences that contained between one and five polymorphic sites) representing two distinct assemblages of Giardia (recognized in the current literature) were defined, of which 13 were new records. One dominant sequence type (with accession no. GQ444447, representing a genotype within assemblage A) has been detected previously in humans and is thus considered to be of zoonotic potential. (Nucleotide sequences reported in this article are available in the GenBank database under accession nos. GQ444447-GQ444451 and GQ444454-GQ444462).

A theoretical study to establish the relationship between the three-dimensional structure of triose-phosphate isomerase of Giardia duodenalis and point mutations in the respective gene.

Predicting how point mutations in genes alter the tertiary and quarternary structure of proteins is central to a number of areas of molecular biology and has implications in relation to the function and evolution of molecules. In the present study, we theoretically assessed the effects of 20 point mutations detected previously in a region of the triose-phosphate isomerase gene (tpi) of the protozoan Giardia duodenalis on the three-dimensional structure of the 'wild-type' protein (TPI). Amino acid substitutions arising from codon variations were mainly located at surface-accessible sites or in hydrophobic pockets of TPI. None of the substitutions was predicted to exert a significant change to the fold or functionality of the enzyme, with the exception of one alteration (Arg100). Almost all substitutions were either conservative or semi-conservative, and retained or even improved the expected stability of the fold. Overall, the findings provide support for the "neutral theory", which contends that evolution at the molecular level is not solely shaped by "Darwinian selection but also by random drift of selectively neutral or nearly neutral mutants".

Genetic classification of Echinococcus granulosus cysts from humans, cattle and camels in Libya using mutation scanning-based analysis of mitochondrial loci.

We genetically classified Echinococcus granulosus from humans, cattle and camels in Libya utilizing DNA regions (designated pcox1 and pnad1) within the cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase 1 (nad1) mitochondrial genes, respectively. Polymerase chain reaction (PCR)-based single-strand conformation polymorphism (SSCP) analysis of pcox1 and pnad1 amplicons derived from genomic DNA samples from individual cysts (n = 176) revealed four distinct electrophoretic profiles for each locus. Direct sequencing of selected amplicons representing each of these profiles defined four different sequence types for each locus, which were present in five different combinations (designated haplotypes A-E) amongst all 176 isolates. Phylogenetic analysis of concatenated sequence data for these five haplotypes, together with a range of well-defined reference sequences, inferred that all cyst isolates from humans (n = 55) and a small number from cattle (13% of 38) belonged to the G1-G3 complex of E. granulosus (or E. granulosus sensu stricto), whereas most (87%) cysts from cattle and all 83 of them from camels were linked to the G6-G10 complex (or Echinococcus canadensis). The present study provides a foundation for future large-scale studies of the epidemiology and ecology of E. granulosus in Libya and other African countries.

Two new species of flukes (Digenea: Bucephalidae: Prosorhynchinae) from the western Moray Gymnothorax woodwardi (Anguilliformes: Muraenidae) from off western Australia, with replacement of the pre-occupied generic name Folliculovarium Gu & Shen, 1983.

Two new species of bucephalid trematodes are described from the rectum and intestine of the western moray eel Gymnothorax woodwardi McCulloch (Anguilliformes: Muraenidae: Muraeninae) off Point Peron in Western Australia. Dollfustrema gibsoni n. sp. is distinguished by body size, a pharynx that is intertesticular and level (latero-dextrally) with the anterior portion of the cirrus-sac, an ovary positioned dextrally to the testes and slightly anterior to (in part) the anterior testis, a uterus that extends anteriorly to the vitelline follicles but not to the level of the rhynchus, and vitelline follicles that form a confluent arc anterior to the gonads. Muraenicola nom. nov. is proposed as a replacement generic name for the pre-occupied Folliculovarium Gu & Shen 1983 nec Singh & Sinha 1981. Muraenicola botti n. sp. is distinguished from its congeners by body size, the size of the cirrus-sac (relative to body size), and in possessing tegumental spines, testes that are oblique (rather than in tandem) and eight ovarian lobes. It differs further in having an intestinal caecum that extends anteriorly to the level of the vitelline follicles and in the position of the pharynx and cirrus-sac relative to each other (lateral in part) as well as to the gonads. M. botti n. sp. also lacks a metraterm. These are the first reports of these genera from fishes off Australia and from the southern hemisphere.

Two new species of Prosorhynchoides (Digenea: Bucephalidae) from Tylosurus crocodilus (Belonidae) from the great barrier reef and French Polynesia.

We surveyed 14 individuals of Tylosurus crocodilus Péron & Lesueur 1821 (Belonidae) collected from the waters around Lizard Island and Heron Island, Great Barrier Reef, Queensland, Australia, and the waters around Moorea, French Polynesia. We describe two new species of bucephaline trematodes from them, Prosorhynchoides galaktionovi n. sp. and P. kohnae n. sp. They are morphologically distinct from existing Prosorhynchoides spp., with molecular data from 28S and ITS-2 ribosomal DNA, as well as cox1 mitochondrial DNA, further supporting our morphological findings. Neither species has been observed in other belonid fishes. The new species fall into the clade of species of Prosorhynchoides from belonids previously identified in Australian waters. These findings strengthen the observation that groups of bucephaline species have radiated, at least in part, in tight association with host taxa. There are now five species of Prosorhynchoides known from two belonid species in Australian waters. We, therefore, predict further richness in the nine other belonid species present.

Genetic characterization of Cryptosporidium parvum from calves by mutation scanning and targeted sequencing--zoonotic implications.

This study explored the genetic make-up of Cryptosporidium in fecal samples from 268 individual calves on pasture-based dairy farms in three regions of Victoria, Australia. An integrated approach, using PCR-coupled single-strand conformation polymorphism, targeted sequencing and phylogenetic analysis, was employed to classify the genetic variants (i.e. genotypes and subgenotypes) of Cryptosporidium parvum present in 124 (46.3%) samples and to infer their zoonotic potential. Genotypic and subgenotypic classification was achieved using a portion of the 60 kDa glycoprotein gene (designated pgp60); specific identity was verified using a region within the small subunit of the nuclear ribosomal RNA (pSSU). Twelve sequence types representing ten distinct subgenotypes were defined within genotype IIa, namely IIaA16G3R1 (n=7), IIaA17G2R1 (1), IIaA18G2R1a (2), IIaA18G2R1b (1), IIaA18G4R1 (1), IIaA19G3R1a (80), IIaA19G3R1b (1), IIaA20G2R1 (9), IIaA20G3R1 (1), IIaA20G4R1 (9), IIaA21G3R1 (1) and IIaA23G3R1 (9), of which IIaA18G2R1b, IIaA18G4R1 and IIaA19G3R1b are new records. All of the subgenotypes, except IIaA16G3R1, IIaA18G4R1 and IIaA20G4R1, have been detected previously in humans and are thus considered to be of zoonotic relevance. (Nucleotide sequences reported in this paper are available in the GenBank database under accession numbers FJ825018-FJ825029).

A C-Terminal Fragment of Chlorotoxin Retains Bioactivity and Inhibits Cell Migration.

Chlorotoxin was originally isolated from the venom of the Israeli scorpion Leiurus quinquestriatus, and has potential as a tumor imaging agent based on its selective binding to tumor cells. Several targets have been suggested for chlorotoxin including voltage-gated chloride channels, and it has been shown to have anti-angiogenic activity and inhibit cell migration. The structure of chlorotoxin is stabilized by four disulfide bonds and contains ß-sheet and helical structure. Interestingly, the reduced form has previously been shown to inhibit cell migration to the same extent as the wild type, but structural analysis indicates that the reduced form of the peptide does not maintain the native secondary structure and appears unstructured in solution. This lack of structure suggests that a short stretch of amino acids might be responsible for the bioactivity. To explore this hypothesis, we have synthesized fragments of chlorotoxin without disulfide bonds. As expected for such small peptides, NMR analysis indicated that the peptides were unstructured in solution. However, the peptide corresponding to the eight C-terminal residues inhibited cell migration, in contrast to the other fragments. Our results suggest that the C-terminal region plays a critical role in the bioactivity of chlorotoxin.

Does selection for growth rate in broilers affect their resistance and tolerance to Eimeria maxima?

Chickens exhibit varied responses to infection with Eimeria parasites. We hypothesise that broilers selected for increased growth rate will show lower resistance and tolerance to a coccidian challenge. 288 chickens of fast (F) or slow (S) growing lines were inoculated with 0 (control), 2500 (low-dose), or 7000 (high-dose) sporulated E. maxima oocysts at 13 days of age in two consecutive rounds. Gain and Intake were measured daily and their values relative to BW at the point of infection were calculated over the pre-patent (days 1-4 post-infection), acute (d5-8 pi), and recovery (d9-12 pi) phases of infection to assess the impact of infection. Levels of plasma carotenoids, vitamins E and A, long bone mineralisation, caecal microbiota diversity indices, and histological measurements were assessed at the acute (d6 pi) and recovery stage (d13 pi). In addition, we measured the levels of nitric oxide metabolites and the number of parasite genome copies in the jejunumat d6pi. In absolute terms F birds grew 1.42 times faster than S birds when not infected. Infection significantly reduced relative daily gain and intake (P < 0.001), with the effects being most pronounced during the acute phase (P < 0.001). Levels of all metabolites were significantly decreased, apart from NO which increased (P < 0.001) in response to infection on d6pi, and were accompanied by changes in histomorphometric features and the presence of E. maxima genome copies in infected birds, which persisted to d13pi. Furthermore, infection reduced tibia and femur mineralisation, which also persisted to d13pi. Reductions in measured variables were mostly independent of dose size, as was the level of parasite replication. The impact of infection was similar for S and F-line birds for all measured parameters, and there were no significant interactions between line x dose size on any of these parameters. In conclusion, our results suggest that line differences in productive performance do not influence host responses to coccidiosis when offered nutrient adequate diets.