Turkey Hemorrhagic Enteritis Virus Can Be Titrated but Not Propagated in Chicken Embryos.

This study aimed to investigate the feasibility of propagating and titrating hemorrhagic enteritis virus (HEV) in chicken embryos. A total of 308 embryonated eggs were used. At 10 days of embryonic age, eggs were inoculated via allantoic sac or chorioallantoic membrane routes with non-heat-treated (live) HEV or heat-treated (dead) HEV or served as negative controls. Allantoic fluid retrieved at 0, 1, 3, 5, and 7 days postinoculation (dpi) was tested for HEV by quantitative PCR. Inoculation with HEV did not cause visible growth impairment or lesions in the chicken embryos. Overall, there was no difference in postinoculation mortality rates among groups sham-inoculated (6/30, 20.0%) or inoculated with live (34/252, 13.4%) or dead (3/ 26, 6.9%) HEV (P = 0.58). The amount of HEV DNA detected in allantoic fluid at 7 dpi in eggs inoculated with live virus was similar to the inoculated dose, indicating that virus propagation in chicken embryos is not efficient. No HEV DNA was detected after 3 dpi in eggs inoculated with dead virus. Inoculation of chicken embryos combined with qualitative PCR can be used for titration of HEV virus stocks and presents a high correlation with in vivo titration using chickens (R2 0.98, P = 0.007). This method may be relevant in countries in which specific-pathogen-free turkeys are unavailable and in which the importation of RP19 cells, the only cell that supports effective propagation of HEV, is not permitted.

El virus de la enteritis hemorrágica de los pavos puede ser titulado pero no propagado en embriones de pollo. Este estudio tuvo como objetivo investigar la viabilidad de propagar y titular al virus de la enteritis hemorrágica de los pavos (con las siglas en inglés HEV) en embriones de pollo. Se utilizaron un total de 308 huevos embrionados. A los 10 días de edad embrionaria, los huevos se inocularon por vía saco alantoideo o por la membrana corioalantoidea con el virus de la enteritis hemorrágica sin tratamiento térmico (vivo) o con un virus de la enteritis hemorrágica con tratamiento térmico (muerto) y algunos sirvieron como controles negativos. El fluido alantoideo recuperado a los cero, uno, tres, cinco y siete días después de la inoculación se analizó para detectar el VHE mediante un método cuantitativo de PCR. La inoculación con el virus de la enteritis hemorrágica no causó daños visibles en el crecimiento ni lesiones en los embriones de pollo. En general, no hubo diferencias en las tasas de mortalidad después de la inoculación entre los grupos con inoculación simulada (6/30, 20.0%) o inoculados con el virus vivo de la enteritis hemorrágica (34/252, 13.4%) o con el virus muerto (3/26, 6.9%) (P=0.58). La cantidad de ADN del virus fue detectada en el fluido alantoideo a los siete días después de la inoculación en huevos inoculados con virus vivos fue similar a la dosis inoculada, lo que indica que la propagación del virus en embriones de pollo no fue eficiente. No se detectó ADN del virus de la enteritis hemorrágica después de tres días después de la inoculación en huevos inoculados con virus muerto. La inoculación de embriones de pollo combinada con el método cuantitativo de PCR se puede utilizar para la titulación de lotes del virus de la enteritis hemorrágica y presenta una alta correlación con la titulación in vivo utilizando pollos (R2 0.98, P = 0.007). Este método puede ser relevante en países en los que no se dispone de pavos libres de patógenos específicos y en los que no se permite la importación de células RP19, la única célula que admite la propagación efectiva del virus de la enteritis hemorrágica.

Nutrient sensing-the key to fungal p53-like transcription factors?

The mammalian tumour suppressor protein, p53, plays an important role in cell cycle control, DNA repair and apoptotic cell death. Transcription factors belonging to the "p53-like" superfamily are found exclusively in the Amorphea branch of eukaryotes, which includes animals, fungi and slime molds. Many members of the p53-like superfamily (proteins containing p53, Rel/Dorsal, T-box, STAT, Runt, Ndt80, and the CSL DNA-binding domains) are involved in development. Two families of p53-like proteins (Ndt80 and CSL) are widespread in fungi as well as animals. The Basidiomycetes and the Ascomycetes have undergone reciprocal loss of the Ndt80 and CSL classes of transcription factors, with the CSL class preserved in only one branch of Ascomycetes and the Ndt80 class found in only one branch of Basidiomycetes. Recent studies have greatly expanded the known functions of fungal Ndt80-like proteins and shown that they play important roles in sexual reproduction, cell death, N-acetylglucosamine sensing and catabolism, secondary metabolism, and production of extracellular hydrolases such as proteases, chitinases and cellulases. In the opportunistic pathogen, Candida albicans, Ndt80-like proteins are essential for hyphal growth and virulence and also play a role in antifungal resistance. These recent studies have confirmed that nutrient sensing is a common feature of fungal Ndt80-like proteins and is also found in fungal CSL-like transcription factors, which in animals is the mediator of Notch signalling. Thus, nutrient sensing may represent the ancestral role of the p53-like superfamily.

Propagation of an Avirulent Turkey Hemorrhagic Enteritis Virus Isolate in Chickens.

A series of studies were undertaken to optimize the propagation of hemorrhagic enteritis virus (HEV) in specific-pathogen-free (SPF) chickens. A total of 562 SPF chickens were orally inoculated with an Australian avirulent HEV isolate of turkey origin at 9, 14, 21, or 28 days of age with 5, 6, 7, or 8 log 10 genomic copies (GC), while 102 chickens served as uninfected controls. No clinical signs were observed in infected chickens. There was an inoculum-dose-dependent increase in the relative spleen and liver weight ( P < 0.01). Relative spleen weight 7 days post-HEV inoculation was up to 85% higher in chickens that were inoculated with 6 to 7 GC compared with controls, with no further increase at higher doses. Relative liver weight increased up to 14% in chickens inoculated with 6 GC, with no further increase. Birds inoculated with a 7 GC dose had a higher frequency of HEV DNA-positive birds (77% to 86%) than birds inoculated with lower doses (33% to 59%; P < 0.01). The most efficient dose for live passage propagation was 7 GC inoculated in 9-to-14-day-old birds, yielding an infection rate of 81%. Livers and spleens from infected birds at all doses were processed to produce a putative vaccine with a final GC recovery in the vaccine material of 8.6 GC/bird. In summary, HEV of turkey origin can be readily propagated in SPF chickens, and conditions to maximize viral retrieval were established.

Role of HxkC, a mitochondrial hexokinase-like protein, in fungal programmed cell death.

Apoptosis is a form of programmed cell death (PCD) that occurs during animal development and is also triggered by a variety of signals including nutrient or oxidative stress, hypoxia, DNA damage, viral infection and oncogenic transformation. Though apoptotic-like PCD also occurs in plants and fungi, genes encoding several of the key players in mammalian apoptosis (p53 and BH-domain proteins) have not been identified in these kingdoms. In this report we investigated whether HxkC, a mitochondrial hexokinase-like protein, and XprG, a putative p53-like transcription factor similar to Ndt80, play a role in programmed cell death in the filamentous fungus Aspergillus nidulans. We show that a mutant lacking HxkC is more sensitive to oxidative stress. Autolysis, a form of fungal programmed cell death triggered by carbon starvation, is accelerated in the hxkCΔ1 mutant but not the hxkCΔ1 xprGΔ1 double mutant. In the absence of nutrient stress, the hxkCΔ1 mutant displays XprG-dependent DNA fragmentation typical of apoptosis and elevated levels of intracellular protease. HxkC and XprG are required for catabolism of N-acetylglucosamine, as in Trichoderma reesei. We show that XprG is present in the nucleus. We conclude that, like mammalian mitochondrial hexokinase, HxkC has anti-apoptotic activity and the XprG transcription factor has a pro-apoptotic role in filamentous fungi.

Extreme Diversity in the Regulation of Ndt80-Like Transcription Factors in Fungi.

The Saccharomyces cerevisiae Ndt80 protein is the founding member of a class of p53-like transcription factors that is known as the NDT80/PhoG-like DNA-binding family. The number of NDT80-like genes in different fungi is highly variable and their roles, which have been examined in only a few species, include regulation of meiosis, sexual development, biofilm formation, drug resistance, virulence, the response to nutrient stress and programmed cell death. The protein kinase Ime2 regulates the single NDT80 gene present in S. cerevisiae. In this study we used a genetic approach to investigate whether the Aspergillus nidulans Ime2 homolog, ImeB, and/or protein kinases MpkC, PhoA and PhoB regulate the two NDT80-like genes (xprG and ndtA) in A. nidulans. Disruption of imeB, but not mpkC, phoA or phoB, led to increased extracellular protease activity and a defect in mycotoxin production similar to the xprG1 gain-of-function mutation. Quantitative RT-PCR showed that ImeB is a negative regulator of xprG expression and XprG is a negative regulator of xprG and ndtA expression. Thus, in contrast to Ime2, which is a positive regulator of NDT80 in S. cerevisiae, ImeB is a negative regulator as in Neurospora crassa. However, the ability of Ndt80 to autoregulate NDT80 is conserved in A. nidulans though the autoregulatory effect is negative rather than positive. Unlike N. crassa, a null mutation in imeB does not circumvent the requirement for XprG or NdtA. These results show that the regulatory activities of Ime2 and Ndt80-like proteins display an extraordinarily level of evolutionary flexibility.

Distinct roles for the p53-like transcription factor XprG and autophagy genes in the response to starvation.

Autophagy and autolysis are two cannibalistic pathways which allow filamentous fungi to obtain nutrients once environmental nutrient sources are exhausted. In Aspergillus nidulans, the effects of mutations in two key autophagy genes, atgA, the ATG1 ortholog, and atgH, the ATG8 ortholog, were compared with mutations in xprG, which encodes a transcriptional activator that plays a key role in autolysis. The anti-fungal drug rapamycin induces autophagy in a range of organisms. Mutations in atgA and atgH did not alter sensitivity to rapamycin, which inhibits growth and asexual spore production (conidiation), indicating that autophagy is not required for rapamycin sensitivity in A. nidulans. In contrast, inhibition of conidiation by rapamcyin was partially suppressed by the xprG1 gain-of-function mutation, indicating that XprG acts in the pathway(s) affected by rapamycin. It was anticipated that the absence of an intact autophagy pathway would accelerate the response to starvation. However, extracellular and intracellular protease production in response to carbon or nitrogen starvation was not increased in the atgAΔ and atgHΔ mutants, and the onset of autolysis was not accelerated. Compared to wild-type strains and the xprGΔ and xprG1 mutants, conidiation of the autophagy mutants was reduced in carbon- or nitrogen-limiting conditions but not during growth on nutrient-sufficient medium. Nuclear localization of the global nitrogen regulator AreA in response to nitrogen starvation was blocked in the xprG2 loss-of-function mutant, but not in the atgHΔ mutant. Conversely, the atgAΔ mutation but not the xprGΔ mutation prevented vacuolar accumulation of GFP-AtgH, a hallmark of autophagy. These results indicate that in A. nidulans there is little interaction between autophagy and autolysis and the two pathways are activated in parallel during starvation.

A p53-like transcription factor similar to Ndt80 controls the response to nutrient stress in the filamentous fungus, Aspergillus nidulans.

The Aspergillus nidulans xprG gene encodes a putative transcriptional activator that is a member of the Ndt80 family in the p53-like superfamily of proteins. Previous studies have shown that XprG controls the production of extracellular proteases in response to starvation. We undertook transcriptional profiling to investigate whether XprG has a wider role as a global regulator of the carbon nutrient stress response. Our microarray data showed that the expression of a large number of genes, including genes involved in secondary metabolism, development, high-affinity glucose uptake and autolysis, were altered in an xprG Δ null mutant. Many of these genes are known to be regulated in response to carbon starvation. We confirmed that sterigmatocystin and penicillin production is reduced in xprG (-) mutants. The loss of fungal mass and secretion of pigments that accompanies fungal autolysis in response to nutrient depletion was accelerated in an xprG1 gain-of-function mutant and decreased or absent in an xprG (-) mutant. The results support the hypothesis that XprG plays a major role in the response to carbon limitation and that nutrient sensing may represent one of the ancestral roles for the p53-like superfamily. Disruption of the AN6015 gene, which encodes a second Ndt80-like protein, showed that it is required for sexual reproduction in A. nidulans.

Prevalence of Marek's disease virus in different chicken populations in Iraq and indicative virulence based on sequence variation in the ecoRI-q (meq) gene.

A cross-sectional survey was conducted in six provinces in southern Iraq to determine the point prevalence of Marek's disease virus (MDV) in different chicken populations followed by sequencing the meq gene for phylogenetic analysis and virulence-associated polymorphisms. A total of 109 samples from unvaccinated flocks were analyzed comprising 52 dust and 30 spleen samples from commercial broiler farms and 27 spleens from local layer chickens purchased in the town markets. The overall prevalence of MDV was 49.5% with no significant differences between provinces (P = 0.08) or sample types (P = 0.89). Prevalence ranged from 36.8% in Karbala and Nasiriyah to 65% in Amarah. The percentages of positive samples were 59.1%, 46.7%, and 48.1% in broiler dust, broiler spleen, and layer spleen, respectively. The overall mean (+/- SEM) Log10 MDV viral copy number per milligram of dust or spleen as determined by quantitative PCR was 1.78 +/- 0.19, with no significant differences between provinces (P = 0.10) or sample types (P = 0.38). In positive samples only, the overall mean was 3.43 +/- 0.18. Sequencing of the meq gene from samples that showed high levels of MDV target in qPCR testing was attempted. Nine samples were sequenced. These sequences were compared with meq sequences of MDVs of different pathotype. All the Iraqi MDVs had a short meq gene of 897 base pairs because of the deletion of 123 bp relative to the reference strain Md5. The Iraqi meq sequences also contained single-nucleotide polymorphisms, resulting in differences in the amino acid sequence. All of the nine Iraqi meq genes encoded two repeats of four-proline sequences. The published negative association between four-proline repeat number and MDV virulence suggests that the Iraqi MDVs are likely to be highly virulent, but this needs to be confirmed by in vivo testing. Taken together, these results indicate that MDV is common in unvaccinated commercial and village chickens in southern Iraq, that there is limited meq gene sequence variation, that all sequenced samples had a short meq with two four-proline repeats, and that this is consistent with a high level of virulence.

Deletion of the C-terminus of polynucleotide phosphorylase increases twitching motility, a virulence characteristic of the anaerobic bacterial pathogen Dichelobacter nodosus.

The Gram-negative anaerobe Dichelobacter nodosus is the causative agent of footrot in sheep. Different strains of D. nodosus cause disease of differing severities, ranging from benign to virulent. Virulent strains have greater twitching motility and secrete proteases that are more thermostable than those secreted by benign strains. We have identified polynucleotide phosphorylase (PNPase) as a putative virulence regulator and have proposed that PNPase expression is modulated by the adjacent integration of genetic elements. In this study, we compared PNPase activity in three virulent and four benign strains of D. nodosus and found that PNPase activity is lower in virulent strains. We disrupted the pnpA gene in three benign D. nodosus strains and two virulent strains and showed that deletion of the S1 domain of PNPase reduced catalytic activity. In all but one case, deletion of the PNPase S1 domain had no effect on the thermostability of extracellular proteases. However, this deletion resulted in an increase in twitching motility in benign, but not in virulent strains. Reconstruction of the pnpA gene in two mutant benign strains reduced twitching motility to the parental level. These results support the hypothesis that PNPase is a virulence repressor in benign strains of D. nodosus.

The intD mobile genetic element from Dichelobacter nodosus, the causative agent of ovine footrot, is associated with the benign phenotype.

The Gram-negative anaerobic pathogen Dichelobacter nodosus is the principal causative agent of footrot in sheep. The intA, intB and intC elements are mobile genetic elements which integrate into two tRNA genes downstream from csrA (formerly glpA) and pnpA in the D. nodosus chromosome. CsrA homologues act as global repressors of virulence in several bacterial pathogens, as does polynucleotide phosphorylase, the product of pnpA. We have proposed a model in which virulence in D. nodosus is controlled in part by the integration of genetic elements downstream from csrA and pnpA, altering the expression of these putative global regulators of virulence. We describe here a novel integrated genetic element, the intD element, which is 32kb in size and contains an integrase gene, intD, several genes related to genes on other integrated elements of D. nodosus, a type IV secretion system and a putative mobilisation region, suggesting that the intD element has a role in the transfer of other genetic elements. Most of the D. nodosus strains examined which contained the intD gene were benign, with intD integrated next to pnpA, supporting our previous observation that virulent strains of D. nodosus have the intA element next to pnpA.

Mutations in genes encoding sorting nexins alter production of intracellular and extracellular proteases in Aspergillus nidulans.

XprG, a putative p53-like transcriptional activator, regulates production of extracellular proteases in response to nutrient limitation and may also have a role in programmed cell death. To identify genes that may be involved in the XprG regulatory pathway, xprG2 revertants were isolated and shown to carry mutations in genes which we have named sogA-C (suppressors of xprG). The translocation breakpoint in the sogA1 mutant was localized to a homolog of Saccharomyces cerevisiae VPS5 and mapping data indicated that sogB was tightly linked to a VPS17 homolog. Complementation of the sogA1 and sogB1 mutations and identification of nonsense mutations in the sogA2 and sogB1 alleles confirmed the identification. Vps17p and Vps5p are part of a complex involved in sorting of vacuolar proteins in yeast and regulation of cell-surface receptors in mammals. Protease zymograms indicate that mutations in sogA-C permit secretion of intracellular proteases, as in S. cerevisiae vps5 and vps17 mutants. In contrast to S. cerevisiae, the production of intracellular protease was much higher in the mutants. Analysis of serine protease gene expression suggests that an XprG-independent mechanism for regulation of extracellular protease gene expression in response to carbon starvation exists and is activated in the pseudorevertants.

Isolation of the Bacteriophage DinoHI from Dichelobacter nodosus and its Interactions with other Integrated Genetic Elements.

The Gram-negative anaerobic pathogen Dichelobacter nodosus carries several genetic elements that integrate into the chromosome. These include the intA, intB, intC and intD elements, which integrate adjacent to csrA and pnpA, two putative global regulators of virulence and the virulence-related locus, vrl, which integrates into ssrA. Treatment of D. nodosus strains with ultraviolet light resulted in the isolation of DinoHI, a member of the Siphoviridae and the first bacteriophage to be identified in D. nodosus. Part of the DinoHI genome containing the packaging site is found in all D. nodosus strains tested and is located at the end of the vrl, suggesting a role for DinoHI in the transfer of the vrl by transduction. Like the intB element, the DinoHI genome contains a copy of regA which has similarity to the repressors of lambdoid bacteriophages, suggesting that the maintenance of DinoHI and the intB element may be co-ordinately controlled.

The interaction of induction, repression and starvation in the regulation of extracellular proteases in Aspergillus nidulans: evidence for a role for CreA in the response to carbon starvation.

In Aspergillus nidulans, production of extracellular proteases in response to carbon starvation and to a lesser extent nitrogen starvation is controlled by XprG, a putative transcriptional activator. In this study the role of genes involved in carbon catabolite repression and the role of protein as an inducer of extracellular protease gene expression were examined. The addition of exogenous protein to the growth medium did not increase extracellular protease activity whether or not additional carbon or nitrogen sources were present indicating that induction does not play a major role in the regulation of extracellular proteases. Northern blot analysis confirmed that protein is not an inducer of the major A. nidulans protease, PrtA. Mutations in the creA, creB and creC genes increased extracellular protease levels in medium lacking a carbon source suggesting that they may have a role in the response to carbon starvation as well as carbon catabolite repression. Analysis of glkA4 frA2 and creADelta4 mutants showed that the loss of glucose signalling or the DNA-binding protein which mediates carbon catabolite repression did not abolish glucose repression but did increase extracellular protease activity. This increase was XprG-dependent indicating that the effect of these genes may be through modulation of XprG activity.

Characterization of regulatory non-catalytic hexokinases in Aspergillus nidulans.

Hexokinases catalyse the first step in glucose metabolism and play a role in glucose sensing in mammals, plants and fungi. We describe a new class of hexokinases that appear to be solely regulatory in function. The Aspergillus nidulans hxkD gene (formerly named xprF) encodes a hexokinase-like protein. We constructed hxkDDelta gene disruption mutants which showed increased levels of extracellular protease in response to carbon starvation. The hxkDDelta mutations are not completely recessive, indicating that the level of the gene product is critical. Transcript levels of hxkD increase during carbon starvation and this response is not dependent on functional HxkD. A gene encoding a second atypical hexokinase (HxkC) was identified. The hxkCDelta gene disruption mutant exhibits a phenotype similar, but not identical, to hxkDDelta mutants. As with hxkD, mutations in hxkC are suppressed by loss-of-function mutations in xprG, which encodes a putative transcriptional activator involved in the response to nutrient limitation. We show that GFP-tagged HxkD was found only in nuclei suggesting a regulatory role for HxkD. GFP-tagged HxkC was associated with mitochondria. Homologs of hxkC and hxkD are conserved in multi-cellular fungi. Genes encoding atypical hexokinases are present in many genome sequence databases. Thus, non-catalytic hexokinases may be widespread.

The Aspergillus nidulans xprG (phoG) gene encodes a putative transcriptional activator involved in the response to nutrient limitation.

The Aspergillus nidulans xprG gene is involved in the regulation of extracellular proteases. A plasmid which complemented the xprG2 mutation was shown to carry the phoG gene, reported to encode an acid phosphatase. Two phoGDelta mutants were constructed and were identical in phenotype to an xprG2 mutant. Null mutants were unable to use protein as a carbon or nitrogen source, have lost a repressible acid phosphatase and have pale conidial color. XprG shows similarity to the Ndt80 transcriptional activator, which regulates the expression of genes during meiosis in Saccharomyces cerevisiae. The xprG1 gain-of-function mutant contains a missense mutation in the region encoding the putative DNA-binding domain. The response to carbon, nitrogen, sulfur, and phosphate limitation is altered in xprG(-) mutants suggesting that XprG is involved in a general response to starvation. Ndt80 may also be involved in sensing nutritional status and control of commitment to meiosis in S. cerevisiae.

Multiple genetically distinct groups revealed among clinical isolates identified as atypical Aspergillus fumigatus.

To investigate whether genetic variants of A. fumigatus are found among clinical isolates, four isolates that were originally identified as poorly sporulating strains of Aspergillus fumigatus were subjected to molecular analysis. DNA sequence analysis of the alkaline protease genes of these isolates showed that each is genetically distinct and each shows substantial variation (7 to 11%) from the A. fumigatus nucleotide sequence. Subsequent morphological examination suggested that all of the isolates could be classified as Aspergillus viridinutans. To clarify the taxonomic status of these four clinical isolates and of two previously identified as atypical A. fumigatus isolates, partial beta-tubulin and 18S rRNA gene sequences were determined. Each of the six atypical strains had a unique beta-tubulin sequence, whereas the sequences of three standard isolates of A. fumigatus, which were included as controls, were identical to the published A. fumigatus beta-tubulin sequence. The very low level of DNA sequence variation detected in standard isolates of A. fumigatus compared with other isolates from members of Aspergillus section Fumigati suggests that it may be a relatively recently evolved species. The 18S rRNA gene of two of the atypical isolates differed from that of A. fumigatus at a single nucleotide position. Phylogenetic analyses do not support the classification of all of these isolates as A. viridinutans. Thus, some of these isolates represent new species which are potential opportunistic pathogens.

Thelohania parastaci sp. nov. (Microspora: Thelohaniidae), a parasite of the Australian freshwater crayfish, Cherax destructor (Decapoda: Parastacidae).

Thelohania parastaci sp. nov. infects the Australian freshwater crayfish, Cherax destructor. Data on morphology, developmental patterns and sequences from the small subunit (SSU) and internal transcribed spacer (ITS) regions of the ribosomal DNA (rDNA) of T. parastaci sp. nov. are described. The ultrastructural features of different life cycle stages are very similar to those of the European crayfish parasite Thelohania contejeani. T. parastaci sp. nov. exhibits simultaneous dimorphic sporogony in muscle tissue. Meronts, sporonts and spores are found in muscle tissue, within haemocytes in the hepatopancreas, and in the intestinal wall of infected crayfish. T. parastaci sp. nov. shows 92% sequence identity with T. contejeani and only 67% sequence identity with the fire ant pathogen T. solenopsae, when SSU rDNA sequences are compared. Analysis of SSU rDNA and ITS sequences of T. parastaci sp. nov. from crayfish from Victoria, Western Australia, and New South Wales indicate that the parasite has a wide geographical distribution in Australia.

Thelohania montirivulorum sp. nov. (Microspora: Thelohaniidae), a parasite of the Australian freshwater crayfish, Cherax destructor (Decapoda: Parastacidae): fine ultrastructure, molecular characteristics and phylogenetic relationships.

Thelohania montirivulorum sp. nov., a new species of microsporidian parasite, was found in a highland population of the Australian yabby, Cherax destructor. Data are presented on fine ultrastructure, developmental morphology and DNA sequence of the small subunit ribosomal DNA (SSU rDNA) and internal transcribed spacer region. The phylogenetic relationships of T. montrivulorum sp. nov. and other crayfish parasites in the genus Thelohania, based on the SSU rDNA sequence, are investigated. Fine ultrastructure, patterns of sporogony and SSU rDNA sequence similarities indicate T. montirivulorum sp. nov. is congeneric with T. parastaci, a parasite of lowland populations of C. destructor, and with T. contejeani, a parasite of European freshwater crayfish. SSU rDNA data suggests Thelohania species found in crustacean hosts are more closely related to the Vairimorpha/ Nosema clade of species from insect and crustacean hosts than to the fire ant parasites, T. solenopsae and Thelohania sp.

Ultrastructural characteristics and small subunit ribosomal DNA sequence of Vairimorpha cheracis sp. nov., (Microspora: Burenellidae), a parasite of the Australian yabby, Cherax destructor (Decapoda: Parastacidae).

This is the first record of a species of Vairimorpha infecting a crustacean host. Vairimorpha cheracis sp. nov. was found in a highland population of the Australian freshwater crayfish, Cherax destructor. The majority of spores and earlier developmental stages of V. cheracis sp. nov. were found within striated muscle cells of the thorax, abdomen, and appendages of the crayfish. Only octosporoblastic sporogony within sporophorous vesicles (SPVs) was observed. Diplokaryotic sporonts separated into two uninucleate daughter cells, each of which gave rise to four sporoblasts in a rosette-shaped plasmodium, so that eight uninucleate spores were produced within the persistent ovoid SPV. Ultrastructural features of stages in the octosporoblastic sequence were similar to those described for Vairimorpha necatrix, the type species. Mature spores were pyriform in shape and averaged 3.4x1.9 microm in dimensions. The anterior polaroplast was lamellar in structure, and the posterior polaroplast vesicular. The polar filament was coiled 10-12 times, lateral to the posterior vacuole. The small subunit ribosomal DNA (SSU rDNA) of V. cheracis sp. nov. was sequenced and compared with other microsporidia. V. cheracis sp. nov. showed over 97% sequence identity with Vairimorpha imperfecta and five species of Nosema, and only 86% sequence identity with V. necatrix. The need for a taxonomic revision of the Nosema/Vairimorpha group of species is discussed.

Analysis of sequences flanking the vap regions of Dichelobacter nodosus: evidence for multiple integration events, a killer system, and a new genetic element.

Dichelobacter nodosus is the causative agent of ovine footrot. The vap regions of the D. nodosus genome may have arisen by the integration of a genetic element and may have a role in virulence. The virulent D. nodosus strain A198 has multiple copies of the vap regions. In the present study, sequences to the left and right of vap regions 1, 2 and 3 of strain A198 were analysed by Southern blotting and DNa sequencing. The results suggest that vap regions 1 and 2 rose by independent integration events into different tRNA genes. The discovery of a second integrase gene (intB), a gene with similarity to bacteriophage repressor proteins (regA), and a gene similar to an ORF from a conjugative transposon (gepA), suggests that a second genetic element, either a bacteriophage or a conjugative transposon, is integrated next to vap region 3 in the D. nodosus genome. The arrangement of intB and the vap regions in three other virulent strains and one benign strain was determined using using Southern blotting and PCR. One strain, H1215, contained vapE' and not vapE, and thus resembles vap region 3, suggesting that vap region 3 also may have arisen by an independent integration event. In all strains, a copy of intB was found next to the vap regions. The vap regions contain two genes, vapA and toxA, with similarity to the hig genes of the killer plasmid Rts1. Evidence is presented that vapA and toxA have a similar function in D. nodosus.