Spotlight on avian pathology: red mite, a serious emergent problem in layer hens.

Dermanyssus gallinae, the poultry red mite, is currently the most important ectoparasite of the egg laying industry worldwide with an expanding global prevalence. As a blood-feeder, it causes anaemia and severe welfare issues to the hens and it is a major cause of economic losses. It is also a vector for Salmonella species, avian influenza and potentially for other vector-borne pathogens. Paradoxically, there is a notable lack of funding for research into poultry red mite and an urgent need for effective and safe control strategies, sustainable therapies, prophylactics and integrated pest management.

Eimeria species occurrence varies between geographic regions and poultry production systems and may influence parasite genetic diversity.

Coccidiosis is one of the biggest challenges faced by the global poultry industry. Recent studies have highlighted the ubiquitous distribution of all Eimeria species which can cause this disease in chickens, but intriguingly revealed a regional divide in genetic diversity and population structure for at least one species, Eimeria tenella. The drivers associated with such distinct geographic variation are unclear, but may impact on the occurrence and extent of resistance to anticoccidial drugs and future subunit vaccines. India is one of the largest poultry producers in the world and includes a transition between E. tenella populations defined by high and low genetic diversity. The aim of this study was to identify risk factors associated with the prevalence of Eimeria species defined by high and low pathogenicity in northern and southern states of India, and seek to understand factors which vary between the regions as possible drivers for differential genetic variation. Faecal samples and data relating to farm characteristics and management were collected from 107 farms from northern India and 133 farms from southern India. Faecal samples were analysed using microscopy and PCR to identify Eimeria occurrence. Multiple correspondence analysis was applied to transform correlated putative risk factors into a smaller number of synthetic uncorrelated factors. Hierarchical cluster analysis was used to identify poultry farm typologies, revealing three distinct clusters in the studied regions. The association between clusters and presence of Eimeria species was assessed by logistic regression. The study found that large-scale broiler farms in the north were at greatest risk of harbouring any Eimeria species and a larger proportion of such farms were positive for E. necatrix, the most pathogenic species. Comparison revealed a more even distribution for E. tenella across production systems in south India, but with a lower overall occurrence. Such a polarised region- and system-specific distribution may contribute to the different levels of genetic diversity observed previously in India and may influence parasite population structure across much of Asia and Africa. The findings of the study can be used to prioritise target farms to launch and optimise appropriate anticoccidial strategies for long-term control.

Real-time PCR-based quantification of Eimeria genomes: a method to outweigh underestimation of genome numbers due to PCR inhibition.

Eimeria species parasites can cause the disease coccidiosis in all livestock species, most notably poultry. Traditional diagnostics such as faecal microscopy have now been supplemented by molecular assays including genus-specific and species-specific quantitative polymerase chain reaction (qPCR), although DNA extracted from faecal samples is commonly affected by PCR inhibition. This was confirmed when genomic DNA extracted from chicken faeces inhibited the threshold cycle value of internal positive control (IPC) DNA amplification by 15.33%. Hence, the objective of the present study was to use IPC qPCR to determine PCR inhibition in a series of experimental samples and use the increase in IPC qPCR threshold cycle value as an individual (sample-specific) correction factor for an established 5S rDNA qPCR used to estimate total Eimeria genome numbers. IPC-corrected genome counts were correlated with conventional oocyst per gram counts and compared with non-corrected counts, revealing a 0.1769 increase in correlation coefficient to outweigh underestimation of oocyst counts. Though the sample size used in this study is small, this limitation would be offset by the sample-specific correction factor determined using the IPC along with each sample.

EtCRK2, a cyclin-dependent kinase gene expressed during the sexual and asexual phases of the Eimeria tenella life cycle.

EtCRK2, a cyclin-dependent kinase from the coccidian parasite, Eimeria tenella is closely related to eukaryotic cyclin-dependent kinases that regulate progression of the cell cycle and to several cyclin-dependent kinases identified in the Apicomplexa. Northern blot analyses revealed that EtCRK2 is transcribed during both asexual (first-generation schizogony) and sexual (oocyst sporulation) replicative phases of the parasite life cycle. In addition, it appears to be transcriptionally regulated during meiosis. Recombinant EtCRK2 produced in Escherichia coli has kinase activity which is significantly stimulated by the addition of vertebrate cyclin A. This cyclin-dependent kinase may play a significant role in regulating critical cell cycle events during both asexual proliferation and sexual development of the parasite.

TgM2AP participates in Toxoplasma gondii invasion of host cells and is tightly associated with the adhesive protein TgMIC2.

Like other members of the medically important phylum Apicomplexa, Toxoplasma gondii is an obligate intracellular parasite that secretes several classes of proteins involved in the active invasion of target host cells. Proteins in apical secretory organelles known as micronemes have been strongly implicated in parasite attachment to host cells. TgMIC2 is a microneme protein with multiple adhesive domains that bind target cells and is mobilized onto the parasite surface during parasite attachment. Here, we describe a novel parasite protein, TgM2AP, which is physically associated with TgMIC2. TgM2AP complexes with TgMIC2 within 15 min of synthesis and remains associated with TgMIC2 in the micronemes, on the parasite surface during invasion and in the culture medium after release from the parasite plasma membrane. TgM2AP is proteolytically processed initially when its propeptide is removed during transit through the golgi and later while it occupies the parasite surface after discharge from the micronemes. We show that TgM2AP is a member of a protein family expressed by coccidian parasites including Neospora caninum and Eimeria tenella. This phylogenic conservation and association with a key adhesive protein suggest that TgM2AP is a fundamental component of the T. gondii invasion machinery.

EtMIC4: a microneme protein from Eimeria tenella that contains tandem arrays of epidermal growth factor-like repeats and thrombospondin type-I repeats.

Micronemes are specialised secretory organelles that release their proteins by a stimulus-coupled exocytosis that occurs when apicomplexan parasites make contact with target host cells. These proteins play crucial roles in motility and invasion, most likely by mediating adhesion between parasite and host cell surfaces and facilitating the transmission of dynamic forces generated by the parasite actinomyosin cytoskeleton. Members of the TRAP family of microneme proteins are characterised by having extracellular domains containing one or more types of cysteine-rich, adhesive modules, highly-conserved transmembrane regions and cytosolic tails that contain one or more tyrosines, stretches of acidic residues and a single tryptophan. In this paper, we describe a novel member of the TRAP family, EtMIC4, a 218 kDa microneme protein from Eimeria tenella. EtMIC4 contains 31 epidermal growth factor (EGF) modules, 12 thrombospondin type-1 (TSP-1) modules and a highly acidic, proline and glycine-rich region in its extracellular region, plus the conserved transmembrane and cytosolic tail. Like EtMIC1, another TRAP family member from E. tenella, EtMIC4 is expressed in sporozoites and all the merozoite stages of the parasite, suggesting that this parasite has a strong requirement for TSP-1 modules. Unlike the other microneme proteins so far studied in E. tenella, EtMIC4 appears to be found constitutively on the sporozoite surface as well as within the micronemes.

Domains of invasion organelle proteins from apicomplexan parasites are homologous with the Apple domains of blood coagulation factor XI and plasma pre-kallikrein and are members of the PAN module superfamily.

Micronemes are specialised organelles, found in all apicomplexan parasites, which secrete molecules that are essential for parasite attachment to and invasion of host cells. Regions of several microneme proteins have sequence similarity to the Apple domains (A-domains) of blood coagulation factor XI (FXI) and plasma pre-kallikrein (PK). We have used mass spectrometry on a recombinant-expressed, putative A-domain from the microneme protein EtMIC5 from Eimeria tenella, to demonstrate that three intramolecular disulphide bridges are formed. These bridges are analogous to those that stabilise A-domains in FXI and PK. The data confirm that the apicomplexan domains are structural homologues of A-domains and are therefore novel members of the PAN module superfamily, which also includes the N-terminal domains of members of the plasminogen/hepatocyte growth factor family. The role of A-domains/PAN modules in apicomplexan parasites is not known, but their presence in the microneme suggests that they may be important for mediating protein-protein or protein-carbohydrate interactions during parasite attachment and host cell invasion.

Mix and match modules: structure and function of microneme proteins in apicomplexan parasites.

Microneme organelles are found in the apical complex of all apicomplexan parasites and play an important role in the invasion process. The recent identification of microneme proteins from different apicomplexan genera has revealed a striking conservation of structural domains, some of which show functional complementation across species. This supports the idea that the mechanism of host cell invasion across the phylum is conserved not only morphologically, but also functionally at the molecular level. Here, we review and summarize these recent findings.

A microneme protein from Eimeria tenella with homology to the Apple domains of coagulation factor XI and plasma pre-kallikrein.

Microneme organelles are present in all apicomplexan protozoa and contain proteins that are critical for parasite motility and host cell invasion. One apicomplexan-wide family of microneme proteins has been identified with members that are characterised by the possession of thrombospondin type I repeats, conserved adhesive motifs which are implicated in binding to glycosaminoglycan chains. In this paper we describe a micronemal glycoprotein, EtMIC 5, from Eimeria tenella which contains eleven cysteine-rich motifs that have striking similarity to the adhesive Apple (A-) domains of blood coagulation factor XI and plasma pre-kallikrein. EtMIC 5 is confined to an intracellular location in resting sporozoites but is translocated to the parasite surface and secreted into the culture supernatant during parasite infection of MDBK cells. During intracellular replication, the protein is switched off in early schizogony and is then re-expressed within the apical tips of newly formed merozoites. A-domain sequences were also found in microneme proteins from Sarcocystis muris and Toxoplasma gondii and in a protein of unknown localisation from Eimeria acervulina. These studies suggest that A-domain containing proteins may comprise a novel apicomplexan-wide family of microneme adhesins.

A survey of genes in Eimeria tenella merozoites by EST sequencing.

A study of about 500 expressed sequence tags (ESTs), derived from a merozoite cDNA library, was initiated as an approach to generate a larger pool of gene information on Eimeria tenella. Of the ESTs, 47.7% had matches with entries in the databases, including ribosomal proteins, metabolic enzymes and proteins with other functions, of which 14.3% represented previously known E. tenella genes. Thus over 50% of the ESTs had no significant database matches. The E. tenella EST dataset contained a range of highly abundant genes comparable with that found in the EST dataset of T. gondii and may thus reflect the importance of such molecules in the biology of the apicomplexan organisms. However, comparison of the two datasets revealed very few homologies between sequences of apical organelle molecules, and provides evidence for sequence divergence between these closely-related parasites. The data presented underpin the potential value of the EST strategy for the discovery of novel genes and may allow for a more rapid increase in the knowledge and understanding of gene expression in the merozoite life cycle stage of Eimeria spp.

PCR identification of chicken Eimeria: a simplified read-out.

A polymerase chain reaction (PCR) assay, based on the amplification of internal transcribed spacer 1 (ITS1) regions of ribosomal DNA, was developed for the chicken coccidian species Eimeria maxima, E. mitis and E. praecox. Thus, taking into account our previous work, a complete set of ITS1-based, species-specific primers for the detection and discrimination of all seven Eimeria species that infect the domestic fowl is now available. ITS1 primers for each of these seven species of Eimeria were also used as capture probes in a paper chromatography assay (PACHA). The addition of PACHA to the PCR assay provided a faster, more simplified read-out compared to staining of amplified bands in an agarose gel with ethidium bromide.

Inhibition of the development of Eimeria tenella in cultured bovine kidney cells by a soluble factor produced by peripheral blood lymphocytes from immune chickens.

The intracellular development of Eimeria tenella sporozoites in in vitro cultured Madin-Darby Bovine Kidney (MDBK) cells was inhibited when parasite-infected MDBK cells were incubated with peripheral blood lymphocytes (PBL) from infected chickens. The inhibition mediated by PBL was quantified by [3H]uracil uptake and increased during the course of a series of oral infections of chickens with E. tenella. This was mirrored by the development of immunity in these birds, as assessed by counting the oocyst output following each re-infection. Similar levels of inhibition were observed using PBL from 3 inbred lines of chickens which differ in their relative susceptibility to infection with E. tenella, indicating that the genetic background of the host does not influence the production of this inhibitory activity. The inhibition could be transferred to freshly infected MDBK cells using supernatants prepared from parasite-infected monolayers incubated for 48 h with PBL from immune chickens. However, there was no inhibition using either supernatants from infected MDBK cells incubated with PBL from uninfected chickens, or supernatants from uninfected MDBK cells incubated with PBL from immune chickens. Experiments using Transwell plates showed that direct contact of PBL from immune birds with infected MDBK monolayers was not required to produce supernatants with inhibitory activity. Thus production of soluble inhibitory factor(s) by PBL from immune chickens can be specifically induced by soluble antigens present in the culture media of parasite-infected MDBK cells. These factors inhibit the intracellular development of sporozoites in in vitro culture.

Transient expression of beta-galactosidase in differentiating sporozoites of Eimeria tenella.

A transient transfection system has been developed for a member of the Apicomplexa, Eimeria tenella, using beta-galactosidase (betagal) from Escherichia coli as the reporter enzyme. Successfully expressed constructs contained sequences of the E. tenella microneme gene Etmic-1 fused to the coding region of lacZ. Transfectants expressing betagal were able to invade host cells and proceed through part of the life-cycle, forming schizonts from which merozoites were released. This indicated that transfectants could differentiate at least to first generation schizonts. However, this differentiation was delayed compared with unelectroporated sporozoites by approximately 15 h. Some merozoites arising from transfected sporozoites also expressed betagal. These results are encouraging for the development of a stable transfection system for E. tenella, using betagal as a reporter enzyme.

Development of a diagnostic PCR assay for the detection and discrimination of four pathogenic .Eimeria species of the chicken.

We describe a polymerase chain reaction (PCR)-based assay for the detection, identification and differentiation of pathogenic species of .Eimeria in poultry. The internal transcribed spacer 1 (ITS1) regions of ribosomal DNA (rDNA) from .Eimeria acervulina, E. brunetti, E. necatrix and .E. tenella were sequenced and regions of unique sequences identified. Four pairs of oligonucleotide primers, each designed to amplify the ITS1 region of a single .Eimeria species, were synthesised for use in the PCR assay. In tests on purified genomic DNA from all seven species of .Eimeria that infect the chicken, each of the four primer pairs amplified the ITS1 region from only their respective target species. The robustness of the approach was further demonstrated by the amplification of specific DNA fragments from tissues of experimentally infected animals and from oocysts recovered from field samples. We conclude that the ITS1 regions of .Eimeria species contain sufficient inter-specific sequence variation to enable the selection of primers that can be applied in PCR analyses to detect and differentiate between species. In future work they may provide excellent markers for epidemiological studies.

Sequence, expression and localization of calmodulin-domain protein kinases in Eimeria tenella and Eimeria maxima.

We have isolated and sequenced cDNA clones from Eimeria tenella and Eimeria maxima which encode proteins that share homology with a recently described family of calmodulin-domain protein kinases. The primary sequence data show that each of the protein kinases can be divided into 2 main functional domains-an amino-terminal catalytic domain typical of serine/threonine protein kinases and a carboxy-terminal domain homologous to calmodulin, which is capable of binding calcium ions at 4 'EF-hand' motifs. Expression of the E. tenella calmodulin-domain protein kinase (EtCDPK) increased towards the end of oocyst sporulation, as judged by Northern and Western blotting, and indirect immunofluorescent antibody labelling showed that within a few minutes of adding sporozoites to target host cells in in vitro culture EtCDPK was found to be specifically associated with a filament-like structure that converges at the apical end of the parasite. Once the parasite entered the host cell EtCDPK appeared to be left on the host cell membrane at the point of entry, indicating a brief yet specific role for this molecule in the invasion of host cells by E. tenella.

Molecular cloning and characterization of a novel acidic microneme protein (Etmic-2) from the apicomplexan protozoan parasite, Eimeria tenella.

A 50 kDa acidic protein, which is found within the microneme organelles of Eimeria tenella sporozoites and merozoites and called E. tenella mic-2, was cloned by immunoscreening of a cDNA expression library. The expression of the protein and its mRNA during the development cycle of the parasite was consistent with de novo formation of microneme organelles during both sporulation and schizogony. Although micronemal origin, indirect immunofluorescent antibody labelling on gluraraldehyde fixed parasites, indicated that the protein was translocated to the sporozoite surface, and, during host cell invasion the protein was focussed at the point of parasite entry and secreted from the host-parasite interface. Either during or just after invasion, Etmic-2 protein became transiently dispersed over the entire surface of the infected cell. One hour after adding sporozoites to host cells, no detectable Etmic-2 protein remained on the host cell surface. A full length cDNA corresponding to Etmic-2 predicted a protein with a classical signal peptide that preceded the mature N-terminus of the protein as determined by direct microsequencing. Regions of the Etmic-2 protein have highly significant similarities to regions within Drosophila melanogaster tropomyosin II and within two known substrates of the cellular regulatory enzyme protein kinase C.

Primary structure of a BiP homologue in Eimeria spp.

cDNA clones for homologues of a molecular chaperone of the endoplasmic reticulum called the immunoglobulin heavy-chain binding protein (BiP) have been isolated from Eimeria maxima and E. tenella sporozoite cDNA libraries. The E. tenella cDNA clone is of full length and has a predicted N-terminal signal sequence of approximately 30 amino acids and a C-terminal tetrapeptide sequence (His-Asp-Glu-Leu) for retention in the lumen of the endoplasmic reticulum.