Conservation of proteins involved in oocyst wall formation in Eimeria maxima, Eimeria tenella and Eimeria acervulina

Vaccination with proteins from gametocytes of Eimeria maxima protects chickens, via transfer of maternal antibodies, against infection with several species of Eimeria. Antibodies to E. maxima gametocyte proteins recognise proteins in the wall forming bodies of macrogametocytes and oocyst walls of E. maxima, Eimeria tenella and Eimeria acervulina. Homologous genes for two major gametocyte proteins - GAM56 and GAM82 - were found in E. maxima, E. tenella and E. acervulina. Alignment of the predicted protein sequences of these genes reveals that, as well as sharing regions of tyrosine richness, strong homology exists in their amino-terminal regions, where protective antibodies bind. This study confirms the conservation of the roles of GAM56 and GAM82 in oocyst wall formation and shows that antibodies to gametocyte antigens of E. maxima cross-react with homologous proteins in other species, helping to explain cross-species maternal immunity.     

A molecular phylogeny of thermophilic fungi

Sequences from 86 fungal genomes and from the two outgroup genomes Arabidopsis thaliana and Drosophila melanogaster were analyzed to construct a robust molecular phylogeny of thermophilic fungi, which are potentially rich sources of industrial enzymes. To provide experimental reference points, growth characteristics of 22 reported thermophilic or thermotolerant fungi, together with eight mesophilic species, were examined at four temperatures: 22 °C, 34 °C, 45 °C, and 55 °C. Based on the relative growth performances, species with a faster growth rate at 45 °C than at 34 °C were classified as thermophilic, and species with better or equally good growth at 34 °C compared to 45 °C as thermotolerant. We examined the phylogenetic relationships of a diverse range of fungi, including thermophilic and thermotolerant species, using concatenated amino acid sequences of marker genes mcm7, rpb1, and rpb2 obtained from genome sequencing projects. To further elucidate the phylogenetic relationships in the thermophile-rich orders Sordariales and Eurotiales, we used nucleotide sequences from the nuclear ribosomal small subunit (SSU), the 5.8S gene with internal transcribed spacers 1 and 2 (ITS 1 and 2), and the ribosomal large subunit (LSU) to include additional species for analysis. These phylogenetic analyses clarified the position of several thermophilic taxa. Thus, Myriococcum thermophilum and Scytalidium thermophilum fall into the Sordariales as members of the Chaetomiaceae, Thermomyces lanuginosus belongs to the Eurotiales, Malbranchea cinnamomea is a member of the Onygenales, and Calcarisporiella thermophila is assigned to the basal fungi close to the Mucorales. The mesophilic alkalophile Acremonium alcalophilum clusters with Verticillium albo-atrum and Verticillium dahliae, placing them in the recently established order Glomerellales. Taken together, these data indicate that the known thermophilic fungi are limited to the Sordariales, Eurotiales, and Onygenales in the Ascomycota and the Mucorales with possibly an additional order harbouring C. thermophila in the basal fungi. No supporting evidence was found for thermophilic species belonging to the Basidiomycota.     

Sustainable coccidiosis control in poultry production: the role of live vaccines

The development of new methods of administering coccidiosis vaccines has facilitated their use in the hatchery and thereby improved prospects for the economic vaccination of broilers. The acquisition of protective immunity to Eimeria species is boosted by further exposure to infection after vaccination. Factors that affect the reproductive efficiency of non-attenuated and attenuated vaccines are considered and the key role that oocyst production plays in establishing and maintaining uniform immunity in a flock of chickens is discussed. In addition to immunisation, a possible advantage to the application of certain vaccines is that their use could repopulate poultry houses with drug-sensitive organisms. Theoretical rotation programmes in which the use of drugs is alternated with that of vaccines are described. Variability of the cross-protective immune response between strains of the same species should be considered during vaccine development and subsequent use. The significance of less common species of Eimeria, not included in all vaccines, also needs to be assessed. An important consideration is the occurrence of pathogens other than Eimeria (such as the bacterium Clostridium) in flocks given coccidiosis vaccines and the methods by which they might be controlled. More research is required into the relationship between bacterial and viral infections of poultry and coccidiosis vaccination. Vaccines need to be developed that are simple to apply and cost effective for use in areas of the world where small-scale poultry production is commonplace. In the near future it is likely that more live vaccines based upon oocysts derived from attenuated strains of Eimeria will be developed but in the longer term vaccines will be based on the selective presentation to the host of specific molecules that can induce protective immunity. This achievement will require significant investment from the private and public sectors, and, if successful, will facilitate the sustainable control of coccidiosis in poultry production.     

Detection, selective isolation and characterisation of Dactylosporangium strains from diverse environmental samples

A culture-independent, nested PCR procedure based on genus-specific oligonucleotide primers detected the presence of members of the genus Dactylosporangium in 14 out of 21 diverse environmental samples. Clones generated from the 14 positive environmental samples formed distinct phyletic lines in the dactylosporangial 16S rRNA gene tree. Presumptive dactylosporangiae were isolated from 7 of these samples using a medium designed to be selective for members of the genus Dactylosporangium, namely Streptomyces Isolation Medium supplemented with gentamicin and antifungal antibiotics. One hundred and two out of 219 representative presumptive dactylosporangiae were considered as authentic members of the genus Dactylosporangium as they gave PCR amplification products using the genus-specific primers and had chemical features typical of dactylosporangiae. Representative of the Dactylosporangium isolates formed distinctive phyletic lines in the dactylosporangial 16S rRNA gene tree, contained the non-ribosomal peptide and type-I polyketide synthase genes and inhibited the growth of Bacillus subtilis, Kocuria rhizophila and Staphylococcus aureus strains. It is evident from these results that the genus Dactylosporangium is underspeciated, widely distributed in natural habitats and is a potentially rich source of novel secondary metabolites.     

Resistance to antiparasitic drugs: the role of molecular diagnosis

Chemotherapy is central to the control of many parasite infections of both medical and veterinary importance. However, control has been compromised by the emergence of drug resistance in several important parasite species. Such parasites cover a broad phylogenetic range and include protozoa, helminths and arthropods. In order to achieve effective parasite control in the future, the recognition and diagnosis of resistance will be crucial. This demand for early, accurate diagnosis of resistance to specific drugs in different parasite species can potentially be met by modern molecular techniques. This paper summarises the resistance status of a range of important parasites and reviews the available molecular techniques for resistance diagnosis. Opportunities for applying successes in some species to other species where resistance is less well understood are explored. The practical application of molecular techniques and the impact of the technology on improving parasite control are discussed.     

Cutaneous leishmaniasis in red kangaroos: isolation and characterisation of the causative organisms

This is the first report of cutaneous leishmaniasis in kangaroos where infection was acquired within Australia. The diagnosis is based on the clinical criteria used for humans, the lesion histopathology, the detection and isolation of parasites from the lesions, and the analysis of the small subunit ribosomal RNA genes using the polymerase chain reaction. Despite a clear indication that the parasites belong to the genus Leishmania, no assignation to a known Leishmania species could be made using these or other less conserved genetic loci such as the non-transcribed spacer of the mini-exon repeat. As is the case in humans, some but not all animals harbouring lesions had antibodies to the isolated parasites or to several other Leishmania species. The isolated parasites displayed two well characterised Leishmania glycoconjugates, the lipophosphoglycan and proteophosphoglycan. They were infectious for mouse macrophages in vitro and established long-term infection at 33 degrees C but not at 37 degrees C. Our findings raise the possibility of transmission to humans, which may be unrecognised and suggest the possibility that imported species of Leishmania could become endemic in Australia.     

Two new species of canaliculate rudists (Dictyoptychidae) from southeastern turkey

Two new species of canaliculate rudist genus Dictyoptychus Douvillé: Dictyoptychus quadrizonalis n. sp. and Dictyoptychus vanensis n. sp., are described from the Maastrichtian limestone block of the ophiolitic unit of the Geva?-Van area, southeastern Turkey. The new species are mainly characterized by the complex structure of the canal layer of the lower valve, and also they seem to be most primitive species of Dictyoptychus and transitional between Dictyoptychus Douvillé and its presumed ancestor, Eodictyoptychus Skelton and El-Asa'ad. Many specimens of the Dictyoptychus paronai (Kühn), allows us to first determination of the upper valve shape and to study also of the lower valve canal layer.     

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.     

A molecular phylogeny and classification of Leptochloa (Poaceae: Chloridoideae: Chlorideae) sensu lato and related genera

Background and Aims

Leptochloa (including Diplachne) sensu lato (s.l.) comprises a diverse assemblage of C₄ (NAD-ME and PCK) grasses with approx. 32 annual or perennial species. Evolutionary relationships and a modern classification of Leptochloa spp. based on the study of molecular characters have only been superficially investigated in four species. The goals of this study were to reconstruct the evolutionary history of Leptochloa s.l. with molecular data and broad taxon sampling.

Methods

A phylogenetic analysis was conducted of 130 species (mostly Chloridoideae), of which 22 are placed in Leptochloa, using five plastid (rpL32-trn-L, ndhA intron, rps16 intron, rps16-trnK and ccsA) and the nuclear ITS 1 and 2 (ribosomal internal transcribed spacer regions) to infer evolutionary relationships and revise the classification.

Key results

Leptochloa s.l. is polyphyletic and strong support was found for five lineages. Embedded within the Leptochloa sensu stricto (s.s.) clade are two Trichloris spp. and embedded in Dinebra are Drake-brockmania and 19 Leptochloa spp.

Conclusions

The molecular results support the dissolution of Leptochloa s.l. into the following five genera: Dinebra with 23 species, Diplachne with two species, Disakisperma with three species, Leptochloa s.s. with five species and a new genus, Trigonochloa, with two species.     

Sarcocystis neurona: molecular characterization of enolase domain I region and a comparison to other protozoa

Sarcocystis neurona causes protozoal myeloencephalitis and has the ability to infect a wide host range in contrast to other Sarcocystis species. In the current study, five S. neurona isolates from a variety of sources, three Sarcocystis falcatula, one Sarcocystis dasypi/S. neurona-like isolate, and one Besnoitia darlingi isolate were used to compare the enolase 2 gene segment containing the domain I region to previously sequenced enolase genes from Neospora caninum, Neospora hughesi, Toxoplasma gondii, Plasmodium falciparum, and Trypanosoma cruzi; enolase 2 segment containing domain I region is highly conserved amongst these parasites of veterinary and medical importance. Immunohistochemistry results indicates reactivity of T. gondii enolase 1 and 2 antibodies to S. neurona merozoites and metrocytes, but no reactivity of anti-enolase 1 to the S. neurona bradyzoite stage despite reactivity to T. gondii bradyzoites, suggesting expression differences between organisms.     

Longitudinal multi-locus molecular characterisation of sporadic Australian human clinical cases of cryptosporidiosis from 2005 to 2008

Cryptosporidium is a gastrointestinal parasite that is recognised as a significant cause of non-viral diarrhea in both developing and industrialised countries. In the present study, a longitudinal analysis of 248 faecal specimens from Australian humans with gastrointestinal symptoms from 2005 to 2008 was conducted. Sequence analysis of the 18S rRNA gene locus and the 60 kDa glycoprotein (gp60) gene locus revealed that 195 (78.6%) of the cases were due to infection with Cryptosporidium hominis, 49 (19.8%) with Cryptosporidium parvum and four (1.6%) with Cryptosporidium meleagridis. A total of eight gp60 subtype families were identified; five C. hominis subtype families (Ib, Id, Ie, If and Ig), and two C. parvum subtype families (IIa and IId). The Id subtype family was the most common C. hominis subtype family identified in 45.7% of isolates, followed by the Ig subtype family (30.3%) and the Ib subtype family (20%). The most common C. parvum subtype was IIaA18G3R1, identified in 65.3% of isolates. The more rare zoonotic IId A15G1 subtype was identified in one isolate. Statistical analysis showed that the Id subtype was associated with abdominal pain (p < 0.05) and that in sporadic cryptosporidiosis, children aged 5 and below were 1.91 times and 1.88 times more likely to be infected with subtype Id (RR 1.91; 95% CI, 1.7-2.89; p < 0.05) and Ig (RR 1.88; 95% CI, 1.10-3.24; p < 0.05) compared to children aged 5 and above. A subset of isolates were also analysed at the variable CP47 and MSC6-7 gene loci. Findings from this study suggest that anthroponotic transmission of Cryptosporidium plays a major role in the epidemiology of cryptosporidiosis in Western Australian humans.     

Phomapyrones from blackleg causing phytopathogenic fungi: isolation, structure determination, biosyntheses and biological activity

The isolation and structure determination of phomapyrones D-G, three 2-pyrones and a coumarin, from a group of isolates of the fungal pathogen Leptosphaeria maculans (Desm.) Ces. et de Not., asexual stage Phoma lingam (Tode ex Fr.) Desm, is reported. As well, phomenin B, infectopyrone, and polanrazines B and C were also obtained for the first time from these isolates. In addition, based on results of incorporations of 13C-labeled acetate and malonate, and deuterated methionine, a polyketide pathway is proposed for the biosyntheses of phomapyrones.     

Beyond barcoding: A mitochondrial genomics approach to molecular phylogenetics and diagnostics of blowflies (Diptera: Calliphoridae)

Members of the Calliphoridae (blowflies) are significant for medical and veterinary management, due to the ability of some species to consume living flesh as larvae, and for forensic investigations due to the ability of others to develop in corpses. Due to the difficulty of accurately identifying larval blowflies to species there is a need for DNA-based diagnostics for this family, however the widely used DNA-barcoding marker, cox1, has been shown to fail for several groups within this family. Additionally, many phylogenetic relationships within the Calliphoridae are still unresolved, particularly deeper level relationships. Sequencing whole mt genomes has been demonstrated both as an effective method for identifying the most informative diagnostic markers and for resolving phylogenetic relationships. Twenty-seven complete, or nearly so, mt genomes were sequenced representing 13 species, seven genera and four calliphorid subfamilies and a member of the related family Tachinidae. PCR and sequencing primers developed for sequencing one calliphorid species could be reused to sequence related species within the same superfamily with success rates ranging from 61% to 100%, demonstrating the speed and efficiency with which an mt genome dataset can be assembled. Comparison of molecular divergences for each of the 13 protein-coding genes and 2 ribosomal RNA genes, at a range of taxonomic scales identified novel targets for developing as diagnostic markers which were 117–200% more variable than the markers which have been used previously in calliphorids. Phylogenetic analysis of whole mt genome sequences resulted in much stronger support for family and subfamily-level relationships. The Calliphoridae are polyphyletic, with the Polleninae more closely related to the Tachinidae, and the Sarcophagidae are the sister group of the remaining calliphorids. Within the Calliphoridae, there was strong support for the monophyly of the Chrysomyinae and Luciliinae and for the sister-grouping of Luciliinae with Calliphorinae. Relationships within Chrysomya were not well resolved. Whole mt genome data, supported the previously demonstrated paraphyly of Lucilia cuprina with respect to L. sericata and allowed us to conclude that it is due to hybrid introgression prior to the last common ancestor of modern sericata populations, rather than due to recent hybridisation, nuclear pseudogenes or incomplete lineage sorting.     

Genetics and evolution of triatomines: from phylogeny to vector control

Triatomines are hemipteran bugs acting as vectors of the protozoan parasite Trypanosoma cruzi. This parasite causes Chagas disease, one of the major parasitic diseases in the Americas. Studies of triatomine genetics and evolution have been particularly useful in the design of rational vector control strategies, and are reviewed here. The phylogeography of several triatomine species is now slowly emerging, and the struggle to reconcile the phenotypic, phylogenetic, ecological and epidemiological species concepts makes for a very dynamic field. Population genetic studies using different markers indicate a wide range of population structures, depending on the triatomine species, ranging from highly fragmented to mobile, interbreeding populations. Triatomines transmit T. cruzi in the context of complex interactions between the insect vectors, their bacterial symbionts and the parasites; however, an integrated view of the significance of these interactions in triatomine biology, evolution and in disease transmission is still lacking. The development of novel genetic markers, together with the ongoing sequencing of the Rhodnius prolixus genome and more integrative studies, will provide key tools to expanding our understanding of these important insect vectors and allow the design of improved vector control strategies.     

Novel natriuretic peptides from the venom of the inland taipan (Oxyuranus microlepidotus): isolation, chemical and biological characterisation

Three natriuretic-like peptides (TNP-a, TNP-b, and TNP-c) were isolated from the venom of Oxyuranus microlepidotus (inland taipan) and were also present in the venoms of Oxyuranus scutellatus canni (New Guinea taipan) and Oxyuranus scutellatus scutellatus (coastal taipan). They were isolated by HPLC, characterised by mass spectrometry and Edman analysis, and consist of 35-39 amino acid residues. These molecules differ from ANP/BNP through replacement of invariant residues within the 17-membered ring structure and by inclusion of proline residues in the C-terminal tail. TNP-c was equipotent to ANP in specific GC-A assays or aortic ring assays whereas TNP-a and TNP-b were either inactive (GC-A over-expressing cells and endothelium-denuded aortic rings) or weakly active (endothelium-intact aortic rings). TNP-a and TNP-b were also unable to competitively inhibit the binding of TNP-c in endothelium-denuded aortae (GC-A) or endothelium-intact aortae (NPR-C). Thus, these naturally occurring isoforms provide a new platform for further investigation of structure-function relationships of natriuretic peptides.     

Biologic, morphologic, and molecular characterisation of Neospora caninum isolates from littermate dogs

Isolation and biologic and molecular attributes of Neospora caninum from three littermate dogs are described. Tissue cysts were confined to the brain and striated muscles. N. caninum was isolated (isolates NC-6, NC-7, and NC-8) in rodents and cell culture that had been inoculated with brain tissue from the dogs. Schizont-like stages reactive with N. caninum antibodies were seen in cell cultures seeded with bradyzoites released from Percoll-isolated N. caninum tissue cysts from the brain of one dog. Tissue cysts were infective orally to mice and gerbils, but not to cats and dogs. The isolates were also identified as N. caninum by PCR and sequence analysis.     

Occurrence, hosts, morphology, and molecular characterisation of Pasteuria bacteria parasitic in nematodes of the family Plectidae

Parasitic bacteria of the genus Pasteuria are reported for three Anaplectus and four identified and several unidentified Plectus species found in eight countries in various habitats. The pasteurias from plectids agree in essential morphological characters of sporangia and endospores as well as in developmental cycle with those of the Pasteuria species and strains described from tylenchid nematodes, but appear to be mainly distinguished from these by absence of a distinct perisporium in the spores and the endospores obviously not being cup- or saucer-shaped. The wide range of measurements and morphological peculiarities of sporangia and endospores suggest that probably several Pasteuria species have to be distinguished as parasites in Plectidae. From an infected juvenile of an unidentified plectid species the 16S rRNA gene sequence of Pasteuria sp. was obtained. Substantial sequence divergence from described Pasteuria species and its phylogenetic position on molecular trees indicate that this Pasteuria sp. could be considered as a new species. Preliminary results of the analysis of DNA phylogeny of Pasteuria spp. and their nematode hosts provide evidence for incongruence of their phylogenetic history and of host switching events during evolution of the bacterial parasites.     

Allometry, sexual dimorphism, and phylogeny: A cladistic analysis of extant African papionins using craniodental data

This study conducts a phylogenetic analysis of extant African papionin craniodental morphology, including both quantitative and qualitative characters. We use two different methods to control for allometry: the previously described narrow allometric coding method, and the general allometric coding method, introduced herein. The results of this study strongly suggest that African papionin phylogeny based on molecular systematics, and that based on morphology, are congruent and support a Cercocebus/Mandrillus clade as well as a Papio/Lophocebus/Theropithecus clade. In contrast to previous claims regarding papionin and, more broadly, primate craniodental data, this study finds that such data are a source of valuable phylogenetic information and removes the basis for considering hard tissue anatomy “unreliable” in phylogeny reconstruction. Among highly sexually dimorphic primates such as papionins, male morphologies appear to be particularly good sources of phylogenetic information. In addition, we argue that the male and female morphotypes should be analyzed separately and then added together in a concatenated matrix in future studies of sexually dimorphic taxa. Character transformation analyses identify a series of synapomorphies uniting the various papionin clades that, given a sufficient sample size, should potentially be useful in future morphological analyses, especially those involving fossil taxa.