Simultaneous one-tube quantification of host and pathogen DNA with real-time polymerase chain reaction

ABSTRACT Phaeocryptopus gaeumannii is a widespread foliar parasite of Douglas-fir. Although normally innocuous, the fungus also causes the defoliating disease Swiss needle cast in heavily infected needles. The extent of P. gaeumannii colonization in Douglas-fir foliage was estimated with real-time quantitative polymerase chain reaction (PCR) using TaqMan chemistry. In order to derive a normalized expression of colonization, both pathogen and host DNA were simultaneously amplified but individually detected by species-specific primers and TaqMan probes labeled with different fluorescent dyes. Detection of host DNA additionally provided an endogenous reference that served as both an internal positive control and adjusted for variation introduced by sample-to-sample differences in DNA extraction and PCR efficiencies. The genes employed for designing the TaqMan probes and primers were beta-tubulin for the pathogen and a LEAFY/FLORICAULA-like gene involved in floral development for the tree host. Both probe/primer sets exhibited high precision and reproducibility over a linear range of 4 orders of magnitude. This eliminated the need to analyze samples in multiple dilutions when comparing lightly with heavily infected needles. Quantification of the fungus within needles was successful as early as 1 month after initial infection. Real-time PCR is the only method currently available to quantify P. gaeumannii colonization early in the first year of the colonization process.     

A new form of raabeia-type actinosporean (Myxozoa) from the oligochaete Uncinais uncinata

In a study of the oligochaete fauna and their actinosporean parasites in three lakes in Algonquin Park, Canada, a novel form of raabeia-type actinosporean was observed in a single specimen of Uncinais uncinata (?ersted) (Naididae). This form differs from those previously described in its small size, and by having caudal processes that gradually widen and terminate with a single prominent branch.     

Uterine leiomyomas in two Vietnamese pot-bellied pigs (Sus scrofa)

Two Vietnamese pot-bellied pigs (Sus scrofa) were euthanatized after they developed abdominal distension. Necropsy of both pigs revealed large myometrial neoplasms and cystic endometrial hyperplasia. Multiple discrete smaller myometrial neoplasms were also observed in one pig; however, distant metastases were not observed in either animal. The tumors were diagnosed as leiomyomas on the basis of histologic examination and immunohistochemistry. This is the first detailed report of uterine leiomyomas in swine, and it is suggested that this diagnosis may become more common as more aging pigs are examined.     

Comparison of ITS profiling,REP- and ERIC-PCR of Salmonella Enteritidis isolates from Poland

Thirty-one Salmonella Enteritidis strains isolated from chickens, broilers and hens were analysed by genotypic typing including REP-PCR. ERIC-PCR and ITS profiling (PCR-ribotyping). Analysis of DNA banding patterns generated by REP-PCR revealed the presence of 22 different genotypes, which were grouped by dendrogram analysis into three distinct lineages (maximum similarity approx. 50%). Each isolate of S. Enteritidis analysed by ERIC-PCR generated an individual DNA pattern. Again, these isolates could be divided into three distinct genomic groups (maximum similarity approx. 60%) by their ERIC-PCR fingerprints. REP- and ERIC-PCR were found to be more discriminatory for typing of S. Enteritidis than ITS profiling. Amplification of the 16S-23S rDNA spacer region gave nine different profiles of DNA, subdivided into two closely related groups by dendrogram analysis. In summary, data obtained by genotyping methods for S. Enteritidis isolates from regions located in the south-west and the central parts of Poland revealed an enormous heterogeneity among analysed samples, and proved that REP- and ERIC-PCR are highly discriminatory techniques, which can be used, in addition to conventional methods, in epidemiological studies of S. Enteritidis infections.     

Haemolytic-uraemic syndrome in a dog

An 11-year-old female German Shepherd dog presented with lethargy and anorexia, which progressed to haemorrhagic vomiting, diarrhoea and seizures. Serum biochemistry and haematology results showed azotaemia and mild thrombocytopaenia. Euthanasia was elected and the dog was submitted for necropsy examination. There were widespread serosal and mucosal petechial and ecchymotic haemorrhages within the abdomen, with ascites and multiple renal infarcts. The renal infarcts were associated with fibrinoid necrosis and thrombosis of inter-lobular arteries and arterioles. These arterial lesions and clinical signs are consistent with haemolytic-uraemic syndrome, which has not previously been reported in dogs in Europe.     

Evaluation of a DNA probe for the detection of Erwinia herbicola strains pathogenic on Gypsophila paniculata

A 7.5-kb DNA fragment carrying genes for indole acetic acid biosynthesis in Erwinia herbicola pv. gypsophilae was used as a DNA probe to detect gall-forming E. herbicola strains. A quick colony hybridization procedure was developed to detect E. herbicola pv. gypsophilae in cuttings of gypsophila, and was compared with ELISA and pathogenicity tests. The sensitivity of the colony hybridization procedure was sufficient to detect less than 100 colony-forming units after enrichment culture. In mixed cultures, with saprophytes associated with the gypsophila plant, about 10⁴ CFU/ml of the pathogen were detectable. The colony hybridization is specific to gall-forming E. herbicola strains, and is relatively easy to perform. The advantages of using the colony hybridization procedure for practical purposes, compared with ELISA and pathogenicity tests, are discussed.