Differentiation between pathogenic serotype 1 isolates of Marek's disease virus and the Rispens CVI988 vaccine in Australia using real-time PCR and high resolution melt curve analysis.

Two real-time PCR assays were developed which enable quantitation and differentiation between pathogenic Australian isolates of Marek's disease virus (MDV) serotype 1 and the serotype 1 vaccine strain Rispens CVI988. The assays are based on a DNA sequence variation in the meq gene between pathogenic and vaccinal MDV1 which has been confirmed by sequencing of 20 Australian field strains of MDV. Complete specificity has been demonstrated in samples containing pathogenic MDV (n=20), Rispens (3 commercial vaccine strains), or both. The limit of detection of both the Rispens-specific and the pathogenic MDV1-specific assays was 10 viral copies/reaction. The tests successfully differentiated and quantified MDV in mixtures of pathogenic and vaccinal Rispens virus. A high resolution melt curve analysis targeting the same SNP used for the real-time PCR assays was also developed which successfully detected sequence variation between Md5, six Australian MDV1 isolates and the three Rispens vaccines. However it was ineffective at differentiating mixtures of pathogenic and vaccinal MDV1. The real-time PCR assays have both diagnostic and epidemiological applications as they enable differentiation and quantitation of Rispens CVI988 and pathogenic MDV1 in co-infected chickens in Australia.

Detection of infectious bronchitis virus strain N1/88 from the oviduct and feces of experimentally infected vaccinated and unvaccinated hens.

Hens were vaccinated during the rearing phase with infectious bronchitis virus (IBV) vaccines commercially available in Australia (Vic S and A3) or left unvaccinated and then challenged with the N1/88 strain of IBV at 30 wk of age. Oviduct and fecal samples were collected at regular intervals after N1/88 challenge. A locked nucleic acid probe-based reverse transcription real-time PCR test was designed and used to detect the IBV strain N1/88 from the oviduct and feces of unvaccinated and vaccinated laying hens. Using a recombinant plasmid standard, the detection limit of the reaction was found to be 100 copies and independent assay runs showed reproducible threshold cycle values. Viral RNA was detected in the oviduct of 12 unvaccinated then challenged hens and viral RNA increased sharply on d 10 and 12 postinfection (p.i.). By contrast, among the hens in the vaccinated group, N1/88 was detectable only in the oviduct of 2 hens at 8 and 12 d p.i. N1/88 challenge. Viral RNA was detected in feces of 2 unvaccinated hens up to 4 wk p.i. and in 1 vaccinated hen up to 3 wk p.i. This shows that rearing phase vaccination lowers the total viral RNA of the strain N1/88, even though this strain shows considerable antigenic and genetic variation from the vaccine strain. This new test will be useful for the rapid identification of the N1/88 strain of IBV from oviduct and fecal samples.

Recovery of Salmonella and Escherichia coli from commercial egg shells and effect of translucency on bacterial penetration in eggs.

This experiment was conducted to study the prevalence of Salmonella and Escherichia coli (E. coli). from the surface of egg shells, egg shell membranes or pores, and internal contents from unwashed eggs collected from commercial caged layer farms in Australia. Egg shell swabs, shell crush and egg internal contents (yolk and albumen) of an individual egg were processed for bacteriological examination. Salmonella spp. were not detected from any of the egg shell surfaces, egg shell crush or egg internal contents. Thirty five E. coli isolates were isolated from the egg shell surface. Ten E. coli strains were also isolated from shell crush. However, the internal contents of eggs appeared to be sterile. Polymerase chain reaction was performed on forty-five E. coli isolates using primers for heat stable enterotoxin genes A and B (STa and STb) and also for colicin V gene (cvaC). STa gene was detected in four E. coli isolates isolated from egg shell surfaces. All the E. coli isolates were negative for STb and cvaC genes. These data provide useful information regarding the prevalence of virulent E. coli and Salmonella spp. on and in unwashed eggs collected from layer farms. These data also suggest that unwashed eggs collected from caged layer farms are unlikely to be sources of Salmonella outbreaks. Egg shell translucency could be due to changes in the mammillary layer and mamillary cores during the early phases of egg shell formation and has the potential to increase the incidence of microcracks in egg shells, and hence, may be responsible for bacterial penetration. There was a significant correlation between egg shell translucency and egg shell penetration by Salmonella Infantis and E coli. Both strains of bacteria were able to penetrate the translucent egg shells even at very low doses. The penetration, however, was hindered in both translucent and non translucent eggs at 4 degrees C, as compared with room temperature which highlights the importance of storage of eggs at refrigerated temperatures.

Effects of infectious bronchitis virus vaccine on the oviduct of hens.

In Australia, currently, all pullets reared for egg production are vaccinated with live attenuated strains of infectious bronchitis virus. Various vaccines and protocols to control this viral disease have been developed, although the severity of the disease varies from place to place and flock to flock. In the present trial, the effects of vaccine strains A3 and Vic S on the oviduct of laying hens were assessed by histopathology, electron microscopy, serology and also by determining the presence and persistence of viral RNA in the oviduct by real time PCR following the experimental infection. Birds were either unvaccinated or vaccinated with both A3 and Vic S and then mock-infected, challenged with the same attenuated strains, either A3 or Vic S. Some respiratory signs were observed, but were mild and short-lived. There was no drop in egg production in any of the groups. However, there was visual loss of shell colour in the unvaccinated hens challenged with the Vic S strain. Mild histopathology was recorded only in terms of lymphocyte infiltration and occasional submucosal oedema in the infundibulum and very mild gland dilatation in the magnum. Microscopic lesions were not recorded in the isthmus, tubular shell gland or shell gland pouch. Cilia loss was not observed in any region of the oviduct using scanning electron microscopy. Both the A3 and Vic S vaccine strains were detected in the oviduct of vaccinated and unvaccinated hens, mainly on the 12th day p.i. These results indicate that the A3 and Vic S strains replicate at a low level in the oviduct without causing significant damage and hence are safe for the oviduct.

LNA probe-based real-time RT-PCR for the detection of infectious bronchitis virus from the oviduct of unvaccinated and vaccinated laying hens.

In the present study, LNA-probe based real-time PCR was designed for the detection and absolute quantification of infectious bronchitis virus (IBV) from the oviduct of unvaccinated and vaccinated hens after IBV challenge. Using a recombinant plasmid standard, the detection limit of the reaction was found to be 10 copies and independent assay runs showed reproducible Ct values. Amongst the unvaccinated hens, the virus could be detected between 6 and 20 days post-infection (p.i.), with a peak of viral load between 10 and 14 days p.i. The virus was also detectable in the oviduct of vaccinated, challenged hens although the viral load was much lower compared to the viral load in the oviduct of unvaccinated, challenged hens. This indicates that rearing phase vaccination can offer significant protection of the fully functional oviduct against a pathogenic strain of IBV. The present test will be useful for the rapid identification of IBV directly from clinical samples. Most vaccination trials investigating the efficacy of vaccines for layer and breeder hens have been conducted based on the respiratory tract response. Evaluation of viral load from the oviduct of vaccinated and unvaccinated hens is an efficient method for assessing oviduct protection in commercial laying hens.

Improved diagnosis of virulent ovine footrot using the intA gene.

Footrot is a mixed bacterial infection of the hooves of sheep. The gram-negative anaerobic bacterium Dichelobacter nodosus is the principal causative agent, with different strains causing diseases of different severity, ranging from benign to virulent. In Australia, in the state of New South Wales (NSW), only virulent footrot is subject to regulatory action, including quarantine. However, it is often difficult to distinguish benign footrot from virulent footrot in the initial stages of infection, or under adverse climatic conditions. The gelatin gel test, which measures the thermostability of secreted bacterial proteases, is the laboratory test most widely used in Australia to aid in the differential diagnosis of footrot. The proteases of virulent strains are, in general, more thermostable than the proteases of benign strains. However, there are some false positives in the gelatin gel test, which may lead to unnecessary quarantine procedures. We used Southern blot analysis on 595 isolates of D. nodosus from 124 farms on which sheep had benign or virulent footrot to test for the presence of the intA gene. We found that for D. nodosus strains which are stable in the gelatin gel test, there is a high correlation between the presence of the intA gene and the ability of the strain to cause virulent footrot. We also developed a PCR-based assay for the rapid detection of intA, which can be used to test DNA extracted from colonies grown on plates, or DNA extracted from cotton swabs of culture plates.

The Aspergillus nidulans xprF gene encodes a hexokinase-like protein involved in the regulation of extracellular proteases.

The extracellular proteases of Aspergillus nidulans are produced in response to limitation of carbon, nitrogen, or sulfur, even in the absence of exogenous protein. Mutations in the A. nidulans xprF and xprG genes have been shown to result in elevated levels of extracellular protease in response to carbon limitation. The xprF gene was isolated and sequence analysis indicates that it encodes a 615-amino-acid protein, which represents a new type of fungal hexokinase or hexokinase-like protein. In addition to their catalytic role, hexokinases are thought to be involved in triggering carbon catabolite repression. Sequence analysis of the xprF1 and xprF2 alleles showed that both alleles contain nonsense mutations. No loss of glucose or fructose phosphorylating activity was detected in xprF1 or xprF2 mutants. There are two possible explanations for this observation: (1) the xprF gene may encode a minor hexokinase or (2) the xprF gene may encode a protein with no hexose phosphorylating activity. Genetic evidence suggests that the xprF and xprG genes are involved in the same regulatory pathway. Support for this hypothesis was provided by the identification of a new class of xprG(-) mutation that suppresses the xprF1 mutation and results in a protease-deficient phenotype.

Analysis of two Aspergillus nidulans genes encoding extracellular proteases.

Characterization of prtADelta mutants, generated by gene disruption, showed that the prtA gene is responsible for the majority of extracellular protease activity secreted by Aspergillus nidulans at both neutral and acid pH. The prtA delta mutation was used to map the prtA gene to chromosome V. Though aspartic protease activity has never been reported in A. nidulans and the prtADelta mutants appear to lack detectable acid protease activity, a gene (prtB) encoding a putative aspartic protease was isolated from this species. Comparison of the deduced amino acid sequence of PrtB to the sequence of other aspergillopepsins suggests that the putative prtB gene product contains an eight-amino-acid deletion prior to the second active site Asp residue of the protease. RT-PCR experiments showed that the prtB gene is expressed, albeit at a low level.

Identification and characterization of a native Dichelobacter nodosus plasmid, pDN1.

The gram-negative anaerobe Dichelobacter nodosus is the primary causative agent of ovine footrot, a mixed bacterial infection of the hoof. We report here the characterization of a novel native plasmid, pDN1, from D. nodosus. Sequence analysis has revealed that pDN1 has a high degree of similarity to broad-host-range plasmids belonging, or related, to Escherichia coli incompatibility group Q. However, in contrast to these plasmids, pDN1 encodes no antibiotic resistance determinants, lacks genes E and F, and hence is smaller than all previously reported IncQ plasmids. In addition, pDN1 belongs to a different incompatibility group than the IncQ plasmids to which it is related. However, pDN1 does contain the replication and mobilization genes that are responsible for the extremely broad host range characteristic of IncQ plasmids, and derivatives of pDN1 replicate in E. coli. In addition, the mobilization determinants of pDN1 are functional, since derivatives of pDN1 are mobilized by the IncPalpha plasmid RP4 in E. coli.

The site-specific integration of genetic elements may modulate thermostable protease production, a virulence factor in Dichelobacter nodosus, the causative agent of ovine footrot.

The gram-negative anaerobe Dichelobacter nodosus is the causative agent of footrot in sheep. The authors have previously characterized two genetic elements, the intA (vap) and intB elements, which integrate into the genome of D. nodosus. In the virulent strain A198 there are two copies of the intA element. One copy is integrated into the 3' end of the tRNA-serGCU gene, close to the aspartokinase (askA) gene, and the second copy is integrated into the 3' end of the tRNA-serGGA gene, next to the polynucleotide phosphorylase (pnpA) gene. In this study, a new genetic element was identified in the benign strain C305, the intC element, integrated into the 3' end of the tRNA-serGCU gene, next to askA. The intC element was found in most D. nodosus strains, both benign and virulent, which were examined, and was integrated into tRNA-serGCU in most strains. Between the askA and tRNA-serGCU genes, a gene (designated glpA), was identified whose predicted protein product has very high amino acid identity with RsmA from the plant pathogen Erwinia carotovora. RsmA acts as a global repressor of pathogenicity in E. carotovora, by repressing the production of extracellular enzymes. In virulent strains of D. nodosus the intA element was found to be integrated next to pnpA, and either the intA or intC element was integrated next to glpA. By contrast, all but one of the benign strains had intB at one or both of these two positions, and the one exception had neither intA, intB nor intC at one position. The loss of the intC element from the virulent strain 1311 resulted in loss of thermostable protease activity, a virulence factor in D. nodosus. A model for virulence is proposed whereby integration of the intA and intC genetic elements modulates virulence by altering the expression of glpA, pnpA, tRNA-serGCU and tRNA-serGGA.

Extreme DNA sequence variation in isolates of Aspergillus fumigatus.

DNA sequence analysis of the alkaline protease gene was used to investigate two Aspergillus fumigatus strains isolated from ostriches (QLD1 and NSW3) and one environmental isolate (FRR 1266) that have shown genetic variation in previous analyses. The results showed that the QLD1 sequence was virtually identical to the published sequences for three human isolates but NSW3 differed in > 6% and FRR 1266 in > 10% of the nucleotides that were analysed. An RFLP assay was designed to determine the distribution of these (and other) genetic variants among environmental and clinical isolates of A. fumigatus.

Development of a method for the identification, using the polymerase chain reaction, of Aspergillus fumigatus isolated from ostriches.

OBJECTIVE: To develop a method for identifying DNA of Aspergillus fumigatus from ostriches, using the polymerase chain reaction (PCR). A fumigatus is the principal causative agent of avian aspergillosis. DESIGN: A biochemical trial. SAMPLE POPULATION: Twelve Aspergillus fumigatus isolates and three other Aspergillus species. PROCEDURE: PCR primers that were based on the sequence of the alkaline protease gene from human isolates of A fumigatus were used. RESULTS: We successfully tested the method on ostrich isolates from five states and showed that the test is specific for A fumigatus. CONCLUSIONS: In most cases the DNA sequence of A fumigatus isolates from ostriches is similar to that of human isolates. DNA sequences vary significantly among A fumigatus isolates, including those from affected ostriches in the same flock. The genetic variation may be used to trace aspergillus infections in ostrich flocks and determine if the disease is transmitted by contact with infected birds.

Identification of a native Dichelobacter nodosus plasmid and implications for the evolution of the vap regions.

Studies on the role of various virulence factors of the ovine pathogen, Dichelobacter nodosus, have suffered from the absence of a mechanism for the introduction of DNA into this organism. As an initial step in the development of genetic methods, we have identified and cloned a native 10-kb plasmid, pJIR896, from a clinical isolate. This plasmid was found to be a circular form of vap region 1/3 that is found in the reference strain, A198. However, pJIR896 lacked the duplicated region present in the A198 sequence and instead contained a 1.7-kb putative insertion sequence, IS1253, which shared similarity to a number of unusual IS elements. A model is proposed for the evolution of vap region 1/3 which involves the integration of a plasmid, such as pJIR896, and subsequent rearrangements resulting from the deletion or transposition of IS1253.

Mutations affecting extracellular protease production in the filamentous fungus Aspergillus nidulans.

The extracellular proteases of Aspergillus nidulans are known to be regulated by carbon, nitrogen and sulphur metabolite repression. In this study, a mutant with reduced levels of extracellular protease was isolated by screening for loss of halo production on milk plates. Genetic analysis of the mutant showed that it contains a single, recessive mutation, in a gene which we have designated xprE, located on chromosome VI. The xprE1 mutation affected the production of extracellular proteases in response to carbon, nitrogen and, to a lesser extent, sulphur limitation. Three reversion mutations, xprF1, xprF2 and xprG1, which suppress xprE1, were characterised. Both xprF and xprG map to chromosome VII but the two genes are unlinked. The xprF1, xprF2 and xprG1 mutants showed high levels of milk-clearing activity on medium containing milk as a carbon source but reduced growth on a number of nitrogen sources. Evidence is presented that the xprE1 and xprG1 mutations alter expression of more than one protease and affect levels of alkaline protease gene mRNA.

A role for bacteriophages in the evolution and transfer of bacterial virulence determinants.

A virulence-associated region in the genome of Dichelobacter nodosus has been shown to contain an integrase gene which is highly related to the integrases of Shigella flexneri phage Sf6 and coliphages P4 and phi R73, together with open reading frames (vapB, C and D) related to genes borne on plasmids in Neisseria gonorrhoeae, Escherichia coli, Actinobacillus actinomycetemcomitans and Treponema denticola. Similar to P4 and phi R73, the vap region is bracketed by putative bacteriophage att sites and is adjacent to a tRNA gene, which suggests that the vap region has been derived by the integration of a bacteriophage, or a plasmid carrying a bacteriophage-related integrase gene. Many similarities in genes and genes clusters encoding virulence determinants have been found in distantly related bacteria. These genes are often located on plasmids in one organism but on the chromosome in others, implying that transmission of the genes has been followed by integration. Thus, the events which have generated the vap regions of D. nodosus may represent a common mechanism for transfer of virulence determinants. A number of genes involved in the virulence of bacterial pathogens are found on integrated bacteriophages, and we suggest that others will prove to be associated with tRNA genes and/or integrase genes derived from bacteriophages. The use of tRNA genes as integration sites for many bacteriophages and plasmids may favour intergeneric transmission, as tRNA genes are highly conserved.

Identification of a gene encoding a bacteriophage-related integrase in a vap region of the Dichelobacter nodosus genome.

Dichelobacter nodosus is the principal causative agent of ovine footrot. Nucleotide (nt) sequences from the D. nodosus genome have been isolated and a series of overlapping lambda clones defining vap (virulence-associated protein) regions 1, 2 and 3 have been reported [Katz et al., J. Bacteriol. 176 (1994) 2663-2669]. In the present study, the limits of the virulence-associated (va) DNA around vap regions 1 and 3 were determined by dot-blot hybridization experiments using plasmid subclones to probe genomic DNA from the D. nodosus virulent strain A198 and the benign strain C305. This va region was found to be approx. 11.9 kb in length, and to be interrupted by a short DNA segment which is also found in the benign D. nodosus strain. Sequence analysis of the entire region revealed an ORF, intA, which is very similar to the integrases of bacteriophages phi R73, P4 and Sf6. Bacteriophages phi R73 and P4 integrate into the 3' ends of tRNA genes, with the integrase genes adjacent to the tRNA genes. A similar arrangement was found in the D. nodosus va region. A 19-bp nt sequence was found to be repeated at the ends of the va region, and may represent the bacteriphage attachment site. These findings suggest that D. nodosus may have acquired these DNA sequences by the integration of a bacteriophage, or an integrative plasmid that contains a bacteriophage-related integrase gene. The high similarity of the D. nodosus integrase to integrases from coliphages suggests that these va sequences may be transferred between distantly related bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterization of an Aspergillus nidulans gene encoding an alkaline protease.

We have cloned an Aspergillus nidulans gene (prtA) encoding an alkaline protease (Alp) by probing an A. nidulans library with a fragment amplified from an Aspergillus oryzae Alp-encoding gene. The nucleotide (nt) sequence of prtA was determined. The structure of prtA is similar to that of the A. oryzae Alp-encoding gene. The prtA gene is composed of four exons which are separated by three introns of 59, 57 and 54 nt. The deduced amino acid sequence of the prtA product shows a high degree of similarity to proteases from A. oryzae, A. fumigatus and A. flavus. Southern blot analysis suggests that only one copy of this gene is found in the genome of A. nidulans. The extracellular proteases of A. nidulans are regulated by nitrogen, carbon and sulfur metabolite repression. The prtA RNA levels were analysed under different nutrient conditions. No prtA transcript was detected in mycelium grown in medium containing glucose, NH4+ and sulfate. However, prtA transcript levels were high in mycelia transferred to medium lacking a nitrogen, carbon or sulfur source.

Genetic organization of the duplicated vap region of the Dichelobacter nodosus genome.

The recombinant plasmid pJIR318 contains a fragment of the Dichelobacter nodosus genome which is associated with virulence. Sequence analysis of the pJIR318 insert has shown that it contains four vap (virulence-associated protein) genes which are homologous to open reading frames found on the Escherichia coli F plasmid and the Neisseria gonorrhoeae cryptic plasmid (M. E. Katz, R. A. Strugnell, and J. I. Rood, Infect. and Immun. 60:4586-4592, 1992). The plasmid pJIR318 hybridizes to three regions of the D. nodosus genome, each of which has now been isolated. Regions 1 and 3 were found to be adjacent in the genome of D. nodosus A198, and the order of the vap genes in vap regions 1 and 2 were shown to be identical. Partial sequence analysis and Southern blot analysis of the vap regions showed that the three regions probably arose by a duplication event(s) followed by insertions and/or deletions. A recombinant plasmid, pJIR749, was isolated from a library of a benign D. nodosus strain, 305. This plasmid contained sequences from both ends of vap region 2. Analysis of pJIR749 showed that the sequences on either side of vap region 2 were separated by 324 bp in the genome of benign strain 305 and that the orientations of the sequences were different. It is clear that a simple insertion or deletion event did not generate the benign and virulent strains studied. A model which describes the evolution of the duplicated vap regions in D. nodosus A198 is presented.

Detection of human interferon-alpha-encoding gene expression.

We have devised a sensitive method based on the polymerase chain reaction (PCR) to detect expression of human interferon-alpha-encoding genes (IFN-A) in general, and specifically, expression of the IFN-A2 or IFN-A4 genes. The utility of the PCR approach was assessed by analysis of cloned IFN-A genes, as well as genomic DNA and mRNA isolated from peripheral blood mononuclear cells. We demonstrate the specific amplification of sequences encoding IFN subtypes IFN-alpha-2 and IFN-alpha-4 from as little as 0.1 pg of IFN-A mRNA. The PCR technique has potential clinical application for the detection of IFN-A expression and, thus, identification of the IFN-alpha subtypes produced, particularly in small biopsy samples or otherwise, where only low numbers of cells are available.

Molecular detection of interferon-alpha expression in multiple sclerosis brain.

The demonstration of intermittent interferonaemia in patients with multiple sclerosis prompted a molecular analysis of brain tissue for expression of interferon-alpha genes. A sensitive method was developed based on the polymerase chain reaction. Primer sets were used that could amplify all interferons-alpha or two particular subtypes, interferon-alpha 2 and interferon-alpha 4. The procedure was successful in detecting expression of interferons-alpha in brain and non-brain tissues in most patients with multiple sclerosis. However, expression was demonstrable also in a similar proportion of patients with other neural diseases, and patients with other illnesses. The data indicate that there can be constitutive expression of interferons-alpha in brain tissue, but the possibility that this becomes amplified in multiple sclerosis was not revealed by this study.