Parasite-specific proliferative responses of chicken spleen cells upon in vitro stimulation with Eimeria tenella antigen.

This study aimed to set up methodology to monitor parasite-specific T-cell activation in vitro using Eimeria tenella-infected chickens. A sonicated E. tenella sporozoite protein preparation was used for the activation of chicken spleen cell cultures. Proliferation assessed by 3H-thymidin incorporation or blast transformation of T-cells assessed by immunofluorescence labelling and flow cytometry were used as read-outs for activation. Results showed that E. tenella-specific proliferation was detected in cultures of spleen cells collected in a 'window' between 8 and 14 days after primary infection. However, due to high variation in proliferative responses between individuals and to high background proliferation, large numbers of observations were needed to obtain significant results. Moreover, the outcome was not improved by increasing the infection dose to chickens or by depletion of T-cell receptor (TCR) γ/δ expressing cells from cultures. An E. tenella-specific blast transformation response was observed for TCRα/ß expressing cells within the same 'window', confirming the identity of the responding cells as classic T-cells. Thus, it is possible to study the kinetics of E. tenella-specific T-cell responses in vitro. However, more in-depth phenotypic identification of the responding T-cells could improve the methodology.

Phylogeny of fish-infecting Calyptospora species (Apicomplexa: Eimeriorina).

There are numerous species of apicomplexans that infect poikilothermic vertebrates, such as fishes, and possess unique morphological features that provide insight into the evolution of this important phylum of parasites. Here, the relationship of the fish-infecting Calyptospora species to other coccidians was investigated based on DNA sequence analysis. Genetic data from the small subunit ribosomal DNA region of the genome were obtained for three of the five nominal species in the genus Calyptospora. Phylogenetic analyses supported a monophyletic lineage sister to a group composed of mostly Eimeria species. The monophyly of Calyptospora species supports the validity of the family Calyptosporidae, but the sister relationship to Eimeria species might also suggest the Eimeriidae be expanded to encompass Calyptospora. The validity of the family Calyptosporidae has been questioned because it is delineated from the Eimeriidae largely based on life cycle characteristics and sporocyst morphology. In general, Eimeria species have a homoxenous life cycle, whereas the type species of Calyptospora is heteroxenous. In the absence of experimental transmission studies, it may be difficult to demonstrate whether all Calyptospora species are heteroxenous. Other distinct morphological characteristics of Calyptospora such as an incomplete sporocyst suture, an apical opening for sporozoite release, a thin veil surrounding sporocysts supported by sporopodia, and a lack of Stieda and sub-Stieda bodies suggest there may be adequate features to delineate these taxa. Even without life cycle data for all species, the morphology and genetic data provide a means to reliably classify Calyptospora species. Placement in either the Calyptosporidae or Eimeriidae is discussed, along with issues relating to the phylogeny of the genus Goussia.

Coccidial infections in commercial broilers: epidemiological aspects and comparison of Eimeria species identification by morphometric and polymerase chain reaction techniques.

The objective of this study was to add to existing knowledge of the epidemiology and the aetiology of coccidial infections in commercial broiler flocks. Polymerase chain reaction (PCR) and morphometric identification of the Eimeria species were compared as means of differentiation in the field samples of faeces and litter. For morphometry, the Eimeria species were categorized into three groups based on lengths of the oocysts. Two random samples of commercial broilers were studied, one during 2000/01 and the other during 2003/04. The prophylactic regime (in-feed narasin), husbandry and methods applied were broadly the same for both subpopulations. Coccidial infection prevalence increased from approximately 45% to approximately 75% during this period, but infection levels (oocysts per gram of faeces) did not significantly change. There were substantial geographical differences in both prevalence and infection levels. A change in Eimeria species profile occurred during the study period. Five Eimeria species were identified at slaughter, by PCR targeting the ITS-1 region of the genome; Eimeria acervulina (100%), Eimeria tenella (77%), Eimeria maxima (25%), Eimeria praecox (10%) and Eimeria necatrix (2%). PCR and morphometric tentative identification were in complete agreement in only 49% of the cases.

A simplified protocol for molecular identification of Eimeria species in field samples.

This study aimed to find a fast, sensitive and efficient protocol for molecular identification of chicken Eimeria spp. in field samples. Various methods for each of the three steps of the protocol were evaluated: oocyst wall rupturing methods, DNA extraction methods, and identification of species-specific DNA sequences by PCR. We then compared and evaluated five complete protocols. Three series of oocyst suspensions of known number of oocysts from Eimeria mitis, Eimeria praecox, Eimeria maxima and Eimeria tenella were prepared and ground using glass beads or mini-pestle. DNA was extracted from ruptured oocysts using commercial systems (GeneReleaser, Qiagen Stoolkit and Prepman) or phenol-chloroform DNA extraction, followed by identification of species-specific ITS-1 sequences by optimised single species PCR assays. The Stoolkit and Prepman protocols showed insufficient repeatability, and the former was also expensive and relatively time-consuming. In contrast, both the GeneReleaser protocol and phenol-chloroform protocols were robust and sensitive, detecting less than 0.4 oocysts of each species per PCR. Finally, we evaluated our new protocol on 68 coccidia positive field samples. Our data suggests that rupturing the oocysts by mini-pestle grinding, preparing the DNA with GeneReleaser, followed by optimised single species PCR assays, makes a robust and sensitive procedure for identifying chicken Eimeria species in field samples. Importantly, it also provides minimal hands-on-time in the pre-PCR process, lower contamination risk and no handling of toxic chemicals.

The current status of the small subunit rRNA phylogeny of the coccidia (Sporozoa).

There is no current comprehensive assessment of the molecular phylogeny of the coccidia, as all recently published papers either deal with subsets of the taxa or sequence data, or provide non-robust analyses. Here, we present a comprehensive and consistent phylogenetic analysis of the available data for the small-subunit ribosomal RNA gene sequence, including a number of taxa not previously studied, based on a Bayesian tree-building analysis and the covariotide model of evolution. The assumptions of the analysis have been rigorously tested, and the benefits and limitations highlighted. Our results provide support for a number of prior conclusions, including the monophyly of the families Sarcocystidae (cyst-forming coccidia) and Eimeriidae (oocyst-forming coccidia), but with bird-host Isospora species in the Eimeriidae and mammal-host species in the Sarcocystidae. However, it is clear that a number of previously reported relationships are dependent on the evolutionary model chosen, such as the placements of Goussia janae, Lankesterella minimia and Caryospora bigenetica. Our results also confirm the monophyly of the subfamilies Toxoplasmatinae and Sarcocystinae, but only some of the previously reported groups within these subfamilies are supported by our analysis. Similarly, only some of the previously reported groups within the Eimeriidae are supported by our analysis, and the genus Eimeria is clearly paraphyletic. There are unambiguous patterns of host-parasite relationship within the coccidia, as most of the well-supported groups have a consistent and restricted range of hosts, with the exception of the Toxoplasmatinae. Furthermore, the previously reported groups for which we found no support all have a diverse range of unrelated hosts, confirming that these are unlikely to be natural groups. The most interesting unaddressed questions may relate to Isospora, which has the fewest available sequences and host-parasite relationships apparently not as straightforward as elsewhere within the suborder.

Biochemical characterisation of the 56 and 82 kDa immunodominant gametocyte antigens from Eimeria maxima.

Two immunodominant gametocyte antigens from Eimeria maxima with M(r) 56 kDa and M(r) 82 kDa have been identified previously as potential candidates for inclusion in a recombinant subunit vaccine against coccidiosis in poultry. Here, these proteins have been biochemically characterised, immunolocalised within the parasite, and sequences for their amino termini determined. These antigens co-purify by affinity chromatography suggesting an interaction with each other. However, separation of the proteins by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in the absence of beta-mercaptoethanol did not reveal the presence of inter-chain disulphide bonds. The true masses of the 56 and 82 kDa antigens are 52450 and 62450 Da, respectively, as determined by mass spectrometry. TX-114 separations suggested that they exist, in part, as soluble proteins within the parasite, and immunolocalisation studies indicated that they were found in the wall forming bodies of macrogametocytes. Separation of the proteins by 2D SDS-PAGE revealed that they are acidic in nature and heterogeneous in charge. Cleavage by neuraminidase and O-glycosidase indicated that the presence of O-linked glycans contributed to some of the charge microheterogeneity of both proteins. The absence of these O-glycans however, did not abolish antibody recognition, suggesting that the development of a recombinant subunit vaccine is possible. A more extensive investigation of the carbohydrate moieties of these proteins revealed that they also possess glucose, fucose, mannose and galactose. There was no evidence for the presence of N-linked glycans. The 56 and 82 kDa antigens were separated from a mixture of proteins in a crude gametocyte lysate by 2D SDS-PAGE, the proteins isolated, and the N-terminus amino acid sequence determined. They showed no homology to each other at the N-terminus, or to any other previously characterised protein. Characterisation of these novel proteins has provided further insights into the molecular mechanisms of gametocyte differentiation in E. maxima.

Evaluation of an enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of canine sarcoptic mange.

Abstract The purpose of this study was to evaluate a serodiagnostic test (enzyme-linked immunosorbent assay; ELISA) for sarcoptic mange in dogs and to characterize the assay antigen, based on the mite Sarcoptes scabiei var. vulpes. The ELISA, applied to sera from 359 dogs suspected of having sarcoptic mange, showed a sensitivity and specificity of 92 and 96%, respectively. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the antigen employed in the ELISA revealed polypeptide bands with molecular weights ranging between 14 and 164 kDa. In Western blot analyses antigens of molecular weights between 62 and 64 kDa dominated. Particularly dominant were antigens of 164 and 147 kDa. These were found to have isoelectrical points in the range of 5.7-6.9. Sera from dogs infected with Cheyletiella sp., Demodex canis, Linognathus setosus and Otodectes cynotis, as well as from dogs allergic to fleas, were negative in the ELISA. Résumé- Le but de cette étude est d'évaluer un test sérologique ELISA pour le diagnostic de la gale sarcoptique chez le chien et de caractériser l'antigène révélateur, extrait de l'acarien Sarcoptes scabiei var. vulpes. Le test ELISA, lors d'une étude conduite avec les sérums de 359 chiens suspects de gale sarcoptique a démontré une sensibilité et une spécificité de 92 et 96%, respectivement. L'électrophorèse en gel polyacrilamide dodécyl sulfate de sodium de l'antigène utilisé dans l'ELISA a révélé des bandes polypeptidiques de poids moléculaire compris entre 14 et 164 kDa. Dans l'analyse en Western blot, les antigènes de poids moléculaire compris entre 62 et 164 kDa étaient les plus abondants, notamment ceux de 164 et 147 kDa. Ces derniers ont des points isoélectriques compris entre 5.7 et 6.9. Les sérums de chiens infectés par des Cheyletiella sp. Demodex canis, Linognathus setosus et Otodectes cynotis, ou par des chiens allergiques aux puces, se sont révélés négatifs en ELISA. [Bornstein, S., Thebo, P., Zakrisson, G. Evaluation of an enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of canine sarcoptic mange (Evaluation d'un test ELISA pour le diagnostic sérologique de la gale sarcoptique canine). Veterinary Dermatology 1996; 7: 21-28.] Resumen El objetivo de este estudio fue el de evaluar una pruba serodiagnóstica (prueba de inmunoadsorción ligada a enzima; ELISA) para la sarna sarcóptica en el perro y caracterizar el antigeno prueba, basado en el ácaro Sarcoptes scabei, var. vulpes. El ELISA, aplicado a sueros de 359 perros sospechosos de padecer sarna sarcóptica, mostró una sensibilidad y especificidad del 92 y 96%, respectivamente. La electroforesis en gel de poliacrilamida dodecil sulfato sódico (SDS-PAGE) del antigeno usado en el ELISA reveló bandas de polipétidos con peso molecular entre 14 y 164 kDa. En el análisis Western blot, predominaron los antigenos de pesos moleculares entre 62 y 164 kDa. Los antigenos entre 164 y 147 kDa fueron especialmente predominantes. Estos tuvieron puntos isoeléctricos entre 5.7 y 6.9. Los sueros de perros infectados por Cheyletiella sp., Demodex canis, Linognathus setosus y Otodectes cynotis, asi como el de perros alérgicos a las pulgas fueron negativos en el ELISA. [Bornstein, S., Thebo, P., Zakrisson, G. Evaluation of an enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of canine sarcoptic mange (Evaluation de una prueba de immunoadsorcion ligada a enzima (ELISA) para el diagnostico serologico de la sarna sarcóptica canina). Veterinary Dermatology 1996; 7: 21-28.] Zusammenfassung- Ziel dieser Studie war, einen Serodiagnostiktest (Enzyme-Linked-Immunosorbent-Assay, ELISA) für Sarkoptesräude des Hundes zu überprüfen und das Testantigen zu charakterisieren, das auf der Milbe Sarcoptes scabiei var. vulpes basiert. Der ELISA-Test, der bei den Sera von 359 Hunden mit Sarkoptesverdacht angewendet wurde, zeigte eine Sensitivität von 92% bzw. 96%. Die Natriumdodecylsul-fatpolyacrylamid-Gelelektrophorese (SDS-PAGE) des Antigen, das im ELISA verwendet wurde, zeigte Polypeptid-Banden mit Molekulargewichten zwischen 14 und 164 kDa. In der Wester-blot-Analyse dominierten Antigene mit einem Molekulargewicht zwischen 62 und 164 kDa. Besonders dominierend waren Antigene von 164 und 147 kDa. Bei diesen stellte man isoelektrische Punkte im Bereich von 5,7 bis 6,9 fest. Die Sera von Hunden, die mit Cheyletiella sp., Demodex canis, Linognathus setosus und Otodectes cynotis infiziert waren, fielen ebenso wie die Hunde mit Allergie auf Flöhe im ELISA negativ aus. [Bornstein, S., Thebo, P., Zakrisson, G. Evaluation of an enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of canine sarcoptic mange (Die Auswertung eines Enzym-Linked-Immunosorbent-Assay (ELISA) für die serologische Diagnose der kaninen Sarkoptesräude). Veterinary Dermatology 1996; 7: 21-28.].