TaqMan Chemistry for Phytophthora ramorum Detection and Quantification, with a Comparison of Diagnostic Methods

ABSTRACT The choice of detection method for phytopathogens can be critically important in determining the success or failure of pest regulation systems. We present an assay for Phytophthora ramorum that uses 5' fluorogenic exonuclease (TaqMan) chemistry to detect and quantify the pathogen from diseased tissue, and include a universal primer and probe set for an internal positive control. This method is sensitive, detecting as little as 15 fg of target DNA when used in a nested design or 50 fg when used in a single round of polymerase chain reaction. None of the 17 other Phytophthora spp. tested was amplified by this assay. A comparison of the nested and non-nested TaqMan assays, and of one other nested assay, showed nested methods to be significantly more sensitive than nonnested and showed that host substrate significantly affected sensitivity of all assays. The nested TaqMan protocol was successfully field-tested; P. ramorum was detected in 255 of 874 plants in California woodlands, whereas the single-round TaqMan protocol detected significantly fewer positive samples. Finally, we documented increases in the quantity of pathogen DNA in Umbellularia californica leaves in initial stages of infection.     

Adaptation to iron deficiency and high pH in evergreen azaleas (<Emphasis Type="Italic">Rhododendron</Emphasis> spp.): potential resources for breeding

The growth of evergreen azaleas (Rhododendron spp.) can be altered by iron (Fe) chlorosis when plants are cultivated in a neutral-alkaline substrate. In this study, morphological and physiological responses to alkalinity and Fe deficiency were evaluated in five diploid Japanese azaleas to assess their potential as resources for breeding. R. obtusum ‘Kirin’, R. indicum ‘Shinsen’, R. × pulchrum ‘Sen-e-oomurasaki’, R. indicum ‘Osakazuki’, and R. ripense were pot cultivated in a peat-based substrate for 10 weeks, in acid and alkaline growing media with both adequate and inadequate Fe nutrition. Plant performance was generally affected by high pH of the substrate, while Fe deficiency by itself influenced few of the evaluated parameters, possibly due to the complex adaptive response mechanisms of these slow growing ornamental shrubs. According to the biochemical and physiological variations recorded on a long period of cultivation, R. indicum ‘Osakazuki’ reported the best performance. This azalea could be a valuable resource for breeders.     

Maize Prolamins Resistant to Peptic-tryptic Digestion Maintain Immune-recognition by IgA from Some Celiac Disease Patients

Maize is used as an alternative to wheat to elaborate foodstuffs for celiac patients in a gluten-free diet. However, some maize prolamins (zeins) contain amino acid sequences that resemble the wheat gluten immunodominant peptides and their integrity after gastrointestinal proteolysis is unknown. In this study, the celiac IgA-immunoreactivity to zeins from raw or nixtamalized grains, before and after peptic/tryptic digestion was evaluated and their possible immunogenicity was investigated by in silico methods. IgA from some celiac patients with HLA-DQ2 or DQ8 haplotypes recognized two alpha-zeins even after peptic/tryptic proteolysis. However, digestion affected zeins after denaturation, reduction, and alkylation, used for identification of prolamins as alpha-zein A20 and A30 by MS/MS sequencing. An in silico analysis indicated that other zeins contain similar sequences, or sequences that may bind even better to the HLA-DQ2/DQ8 molecules compared to the already identified ones. Results concur to indicate that relative abundance of these zeins, along with factors affecting their resistance to proteolysis, may be of paramount clinical relevance, and the use of maize in the formulation and preparation of gluten-free foods must be reevaluated in some cases of celiac disease.     

Urinary cortisol‐creatinine ratio in dogs with hypoadrenocorticism

BackgroundBasal serum cortisol (BSC) ≥2 μg/dL (>55 nmol/L) has high sensitivity but low specificity for hypoadrenocorticism (HA).ObjectiveTo determine whether the urinary corticoid:creatinine ratio (UCCR) can be used to differentiate dogs with HA from healthy dogs and those with diseases mimicking HA (DMHA).AnimalsNineteen healthy dogs, 18 dogs with DMHA, and 10 dogs with HA.MethodsRetrospective study. The UCCR was determined on urine samples from healthy dogs, dogs with DMHA, and dogs with HA. The diagnostic performance of the UCCR was assessed based on receiver operating characteristics (ROC) curves, calculating the area under the ROC curve.ResultsThe UCCR was significantly lower in dogs with HA (0.65 × 10⁻⁶; range, 0.33‐1.22 × 10⁻⁶) as compared to healthy dogs (3.38 × 10⁻⁶; range, 1.11‐17.32 × 10⁻⁶) and those with DMHA (10.28 × 10⁻⁶; range, 2.46‐78.65 × 10⁻⁶) (P < .0001). There was no overlap between dogs with HA and dogs with DMHA. In contrast, 1 healthy dog had a UCCR value in the range of dogs with HA. The area under the ROC curve was 0.99. A UCCR cut‐off value of <1.4 yielded 100% sensitivity and 97.3% specificity in diagnosing HA.Conclusions and Clinical ImportanceThe UCCR seems to be a valuable and reliable screening test for HA in dogs. The greatest advantage of this test is the need for only a single urine sample.     

Assessment of an ELISA method to support surveillance of bovine tuberculosis in Albania

Background

Bovine tuberculosis (bTB) is an important bacterial infectious disease in Albania of concern to animal and human health; its prevalence is poorly documented.

Methods

In this longitudinal study, we tested by ELISA 2661 serum samples, from 154 herds, with the aim of establishing the suitability of this approach to screen the bovine population for bTB. In a follow-on survey of 87 animals in three villages, we assessed the usefulness of the Mycobacterium bovis IDEXX ELISA (IDEXX M. bovis Antibody (Ab) Test. IDEXX Europe B.V P.O. Box 1334, 2130 EK Hoofddorp, The Netherlands) assay by comparing IDEXX results with the results of the single intradermal cervical skin test. Skin tests were performed either after or at the time of collection of blood samples, and therefore cattle were not sensitized by tuberculin before serological testing.

Results

The proportion of herds in which serologically positive cattle were found was 18.2 % (95 % CI, 1.9–25.8 %) and the prevalence of seropositive cattle was 1.4 % (95 % CI, 0.8–2.1 %). In the follow-up study, two of the 87 animals reacted positively to the skin test and two produced inconclusive reactions. No overlap was found between the four animals with positive IDEXX ELISA results and the four animals with non-negative skin test results.

Conclusion

The lack of agreement between the results of the two tests may reflect different elements of the immune response (humoral and cell-mediated immunity). In future, cattle should be sensitized by the intradermal injection of tuberculin 14 days prior to the collection of blood samples, which would then be tested by the Mycobacterium bovis IDEXX ELISA Test in order to determine more accurately the prevalence of infection.

     

Blue-green endoscopy in a dog presenting chronic vomiting-regurgitation

A 2-year-old male Maremma sheepdog presenting with chronic vomiting-regurgitation was examined at the University Veterinary Teaching Hospital, Camerino University. An oesophagogastroscopy with a single blue + green (BG) filter restricting wavelengths from 400 to 550 nm was carried out. A conventional white light endoscopy showed a dilated oesophagus with mildly diffuse erythematous mucosa (more accentuated proximal to the cardia); some portions of the gastric mucosa were covered with fluids and appeared only slightly erythematous. A blue green endoscopy highlighted the oesophageal lesions in dark blue, which made them appear more clearly defined from the remaining mucosa. In the gastric antrum, a small, slightly darker blue roundish area was visible. This area did not show up under the white light endoscopy. A histopathological assessment of biopsy specimens from the distal oesophagus, antrum (including the area highlighted only by BG endoscopy) and gastric body showed chronic-active hyperplastic esophagitis and superficial squamous epithelial dysplasia, while gastric samples showed severe diffuse hyperaemic gastritis of the antrum and superficial diffuse atrophy of the gastric body. The authors believe that the use of a BG endoscopy could be useful in veterinary medicine to increase the diagnostic potential of endoscopic assessment in animals.     

Glyphosate tolerance in maize (Zea mays L.). 2. Selection and characterization of a tolerant somaclone

Summary The progeny of 104 regenerated maize plants were screened for tolerance to the safe broad-spectrum herbicide glyphosate during seed germination and early growth. Seven somaclones showed varying degrees of resistance to the application of the herbicide at 1.2 mM (0.1 kg a.i. in 400 1 ha^-1 of water). Plants capable of a normal growth following treatment with 2.4 mM (0.2 kg ha^-1) glyphosate at the three leaf stage were selfed, and their progeny analyzed. A family able to tolerate the exposure to glyphosate at 2.4 mM was isolated and shown to maintain a photosynthetic rate comparable with control after the application of the herbicide.The selfed progeny of the tolerant somaclone was characterized as to the properties of two targets of glyphosate, the shikimate pathway enzymes 5-enol-pyruvyl-shikimate-3-phosphate (EPSP) synthase and 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase. In vitro tests ruled out the possibility that the tolerance was due to altered forms of these enzymes. Families showed significant variability with regard to EPSP and DAHP synthase levels, measured at different stages during seedling growth; however, not even these traits were correlated with in vivo response to glyphosate. The possible role of other physiological processes in determining the increased tolerance to the herbicide is discussed.Abbreviations DAHP 3-deoxy-D-arabino-heptulosonate-7-phosphate - EPSP 5-enol-pyruvyl-shikimate-3-phosphate - CER carbon-exchange rate - ID₅₀ concentration causing 50% inhibition     

Novel gastric helicobacters and oral campylobacters are present in captive and wild cetaceans

The mammalian gastric and oral mucosa may be colonized by mixed Helicobacter and Campylobacter species, respectively, in individual animals. To better characterize the presence and distribution of Helicobacter and Campylobacter among marine mammals, we used PCR and 16S rDNA sequence analysis to examine gastric and oral samples from ten dolphins (Tursiops gephyreus), one killer whale (Orcinus orca), one false killer whale (Pseudorca crassidens), and three wild La Plata river dolphins (Pontoporia blainvillei). Helicobacter spp. DNA was widely distributed in gastric and oral samples from both captive and wild cetaceans. Phylogenetic analysis demonstrated two Helicobacter sequence clusters, one closely related to H. cetorum, a species isolated from dolphins and whales in North America. The second related cluster was to sequences obtained from dolphins in Australia and to gastric non-H. pylori helicobacters, and may represent a novel taxonomic group. Dental plaque sequences from four dolphins formed a third cluster within the Campylobacter genus that likely represents a novel species isolated from marine mammals. Identification of identical Helicobacter spp. DNA sequences from dental plaque, saliva and gastric fluids from the same hosts, suggests that the oral cavity may be involved in transmission. These results demonstrate that Helicobacter and Campylobacter species are commonly distributed in marine mammals, and identify taxonomic clusters that may represent novel species.