Discrepancy in compliance between the clinical and genetic diagnosis of choroidal hypoplasia in Danish Rough Collies and Shetland Sheepdogs.

Collie eye anomaly (CEA) is a congenital, inherited ocular disorder which is widespread in herding breeds. Clinically, the two major lesions associated with CEA are choroidal hypoplasia (CH) and coloboma, and both lesions are diagnosed based on ophthalmological examination. A 7.8-kb intronic deletion in the gene encoding non-homologous end-joining factor 1 (NHEJ1) has been reported to be the causative mutation underlying CH when present in the homozygous state. In this study, we have investigated the compliance between the clinical and genetic diagnosis of CH in the Danish Rough Collie and Shetland Sheepdog populations. Our results show that the deletion in NHEJ1 is not predictive for CH in the Danish Rough Collie population, whereas the clinical and genetic diagnosis is in accordance with each other in the Shetland Sheepdog population. Based on these results, it can be concluded that the intronic deletion in NHEJ1 is not the causative mutation but, rather, a marker linked to the locus underlying the trait in some populations but linked to both the wild-type and CH-causing locus in most dogs in the Danish Rough Collie population.

Development of a Real-Time Polymerase Chain Reaction Assay for Quantifying Verticillium albo-atrum DNA in Resistant and Susceptible Alfalfa.

ABSTRACT A precise real-time polymerase chain reaction (PCR) assay was developed for quantifying Verticillium albo-atrum DNA. The assay was used in a repeated experiment to examine the relationship between the quantity of pathogen DNA detected in infected leaves and shoots and the severity of Verticillium wilt symptoms in several alfalfa cultivars expressing a range of disease symptoms. Plants were visually inspected for symptoms and rated using a disease severity index ranging from 1 to 5, and the quantity of pathogen DNA present in leaves and stems was determined with real-time PCR. No significant differences in pathogen DNA quantity or disease severity index were observed for experiments or for cultivar-experiment interactions. Significant differences were observed between cultivars for the quantity of pathogen DNA detected with real-time PCR and also for disease severity index ratings. In both experiments, the highly resistant check cultivar Oneida VR had significantly less pathogen DNA, and significantly lower disease severity index ratings than the resistant cultivar Samauri, the moderately resistant cultivar Vernema, and the susceptible check cultivar Saranac. In both experiments, the Spearman rank correlation between the amount of V. albo-atrum DNA detected in leaves and stems with real-time PCR and disease severity index ratings based on visual examination of symptoms was positive (>0.52) and significant (P < 0.0001). These results suggest that resistance to Verticillium wilt in alfalfa is characterized by a reduced colonization of resistant genotypes by the fungus.

A PCR-Based Assay by Sequence-Characterized DNA Markers for the Identification and Detection of Aphanomyces euteiches.

ABSTRACT Polymerase chain reaction (PCR) products were identified and amplified from isolates of Aphanomyces euteiches and A. cochlioides. The products were cloned and sequenced, and the data were used to design pairs of extended PCR primers to amplify sequence-characterized DNA markers. The primer pair OPC7-FS-30 and OPC7-RS-25 amplified a single 1,332-bp product from all isolates of A. euteiches that were not amplified from any other isolates tested. A single 718-bp product was selectively amplified only from isolates of A. cochlioides with the primer pair OPB10-FS-25 and OPB10-RS-25. A. euteiches was detected in roots of several varieties of field-grown peas collected from a root rot trial site. PCR also detected A. euteiches in the organic fraction of field soil samples. Both pairs of extended primers were used in a multiplex reaction to unambiguously discriminate between A. euteiches and A. cochlioides. Both pairs of primers were used in two-step PCR reactions in which annealing and extension was done in a single step at 72 degrees C. This reduced the time required for amplification of the diagnostic PCR product and its resolution by electrophoresis to less than 3 h.

First Report of Red Clover Vein Mosaic Carlavirus Naturally Infecting Lentil.

Lentil (Lens culinaris Medik.) is an important legume crop grown in the dryland Pacific Northwest areas of eastern Washington and Oregon, and northern Idaho. Lentil is highly susceptible to pea enation mosaic enamovirus (PEMV) and bean leafroll luteovirus (BLRV), and infection may result in severe yield losses. Recently, lentil was also found to be infected experimentally with red clover vein mosaic carlavirus (RCVMV) (1). The virus is most commonly transmitted in the Pacific Northwest by the pea aphid (Acyrthosiphon pisum Harris) in a nonpersistent manner. In 1997, cv. Brewer lentil bait plants were planted at the Vegetable Research Farm at Oregon State University to monitor incidence of PEMV and BLRV. Many of the plants developed symptoms typical of PEMV. However, other plants exhibited severe stunting, proliferation of axillary branches, and general chlorosis or death. Bait plants were harvested in August, and 204 random samples were tested for PEMV, RCVMV, BLRV, alfalfa mosaic alfamovirus (AMV), and pea streak carlavirus (PeSV) by standard enzyme-linked immunosorbent assay (ELISA) protocols. Antiserum for RCVMV was made in the Prosser lab against an isolate from chickpea collected in Washington State (1). RCVMV was detected in 76 (34%) of the 204 samples. PEMV, AMV, BLRV, and PeSV were detected in 197 (89.5%), 23 (11.3%), 2 (0.9%), and 0 (0%) of samples, respectively. Results showed that 75/76 of the samples positive for RCVMV were also coinfected with PEMV. Plants infected with RCVMV in the greenhouse also produced mild systemic mosaic symptoms in selected hosts inoculated mechanically, including pea (Pisum sativum L.), chickpea (Cicer arietinum L.), faba bean (Vicia faba L.), and lentil. Lentil and chickpea also showed moderate to severe stunting. Chlorotic local lesions were formed on Chenopodium amaranticolor Coste & Reyn. and C. quinoa Willd. Oligonucleotide primers were designed with sequence data obtained from the Washington isolate of RCVMV (1), and identification of the virus was verified in pea and lentil by polymerase chain reaction (PCR). Primer design of RCV34V and RCV653C targeted a 619-bp fragment located in the viral coat protein gene. Plants testing positive by ELISA yielded PCR products of the expected size when visualized on agarose gels. This is the first report of natural infection of lentil by RCVMV. Reference: (1) R. C. Larsen et al. Phytopathology 87:S56, 1997.

First Report of Bean Leafroll Luteovirus Infecting Pea in Italy.

Approximately 5,000 ha of processing peas (Pisum sativum L) are cultivated annually in the Po River Valley of northern Italy. During the 1998 growing season, affected pea plants in this region were observed that exhibited mild chlorosis and mottling, leaf rolling, and stunting symptoms. High aphid populations and disease levels of nearly 100% were observed in susceptible varieties. Samples from affected fields were analyzed for the presence of bean leafroll virus (BLRV). Nonviruliferous pea aphids (Acyrthosiphon pisum Harris) received a 48-h acquisition access period on symptomatic leaves. Aphids were then transferred to Puget pea and Diana faba bean for a 72-h inoculation access period. Leaf samples were also macerated in 0.05 M potassium phosphate pH 7.4, and inoculated mechanically to pea, faba bean, chickpea (Cicer arietinum L.), Chenopodium quinoa Willd., and C. amaranticolor Coste & Reyn. Symptoms typical of those observed in the original field plants appeared 10 to 14 days after aphid transmissions in both pea and faba bean inoculated with pea aphids. No symptoms were observed in any of the hosts that were inoculated mechanically. Total nucleic acid extracts from the original pea samples, and from leaf tissue of pea and faba bean plants inoculated with aphids, served as templates in reverse-transcriptase polymerase chain reaction assays. Primers BLR-V157 and BLR-C546, which flanked a 400-bp fragment, were designed with available sequence data for the coat protein gene of BLRV (1). An amplification product of the expected size was generated from symptomatic plants but not from healthy controls. Sequence analysis of the cloned fragments revealed a 99% nucleic acid homology with the published sequence for BLRV and an isolate obtained from alfalfa in Washington State (R. Larsen, unpublished). This is the first report of BLRV in Italy. Reference: (1) B. Brill et al. Nucleic Acids Res. 18:5544, 1990.

First Report of Brown Root Rot of Alfalfa Caused by Phoma sclerotioides in Wisconsin.

Brown root rot (BRR) has been associated with winterkill of alfalfa (Medicago sativa L.) in the temperate regions of North America where winters are severe (1). Although suspected, BRR has not been associated with winterkill of alfalfa in the upper Midwestern United States. Alfalfa plants exhibiting symptoms resembling those induced by the causal agent Phoma sclerotioides G. Preuss ex Sacc. were collected from fields in Marinette, Pierce, and Marathon counties in Wisconsin during the spring and early summer of 2003. Symptoms included stunting and decline in 1- to 3-year-old plants that were slow to break dormancy in the early spring. Roots frequently exhibited dark brown lesions or were entirely decayed. Advanced lesions often formed dark bands around the circumference of tap and secondary roots. Beaked pycnidial structures typical of P. sclerotioides were also observed on many samples with advanced lesions. Plants with symptoms of BRR were also observed in Clark, Langlade, Lincoln, Oconto, Shawno, Taylor, and Wood counties. Several lesion areas of tissue on the tap and lateral roots of each sample were excised with a sterile scalpel. Total DNA was extracted using the Fast DNA kit (Bio 101, Carlsbad, CA). In addition, soil samples were collected in the root rhizosphere of symptomatic plants from four fields in two counties. Soil DNA was extracted with the Ultra-Clean DNA soil extraction kit (Mo Bio, Solana Beach, CA). DNA extractions were diluted 1:10 or 1:50, and samples were evaluated for the presence of P. sclerotioides using polymerase chain reaction (PCR)-based sequence-characterized amplified region (SCAR) markers according to the method described previously (4). Amplicons of the expected size (499 bp) were detected from alfalfa roots sampled from Marathon (4 of 4), Marinette (4 of 5), and Pierce (4 of 4) counties but not in roots from healthy controls produced in the greenhouse at Prosser, WA. PCR amplicons were also produced from all field soil samples in Marathon and Marinette counties. Proof of pathogenicity via Koch's postulates for this host-pathogen system was not attempted because of the extensive time period required (1). However, characteristic beaked pycnidia were present, and the pathogen was identified using PCR on DNA from roots symptomatic of BRR. Detection of BRR has been limited in the United States to Wyoming (2), but has been thought to occur in other states with severe winters (3). To our knowledge, this is the first report of P. sclerotioides in Wisconsin. References: (1) J. G. N. Davidson. Brown root rot. Pages 29-31 in: Compendium of Alfalfa Diseases. 2nd ed. D. L. Stuteville and D. C. Erwin, eds. The American Phytopathological Society, St. Paul, MN, 1990. (2) F. A. Gray et al. Pages 27-28 in: Proc. 10th Western Alfalfa Improv. Conf., 1997. (3) C. R. Hollingsworth et al. Can. J Plant Pathol. 25:215, 2003. (4) R. C. Larsen et al. Plant Dis. 86:928, 2002.

A Rapid Method Using PCR-Based SCAR Markers for the Detection and Identification of Phoma sclerotioides: The Cause of Brown Root Rot Disease of Alfalfa.

A rapid technique for identification and detection of Phoma sclerotioides, the causal agent of brown root rot of alfalfa, has been developed using polymerase chain reaction (PCR). Amplification products obtained from random amplified polymorphic DNA (RAPD) reactions were cloned and sequenced, and two extended primer sets were designed from the resulting data that were used to detect sequence-characterized DNA markers. A single 499-bp DNA amplification product was consistently obtained from primers PSB12499 that was specific for 19 isolates of P.sclerotioides but was not produced from Phoma medicaginis or Phoma betae, or from other soilborne pathogens including Aphanomyces euteiches, Rhizoctonia solani, Fusarium oxysporum, Pythium ultimum, or Phytophthora infestans. A 499-bp amplification product was also produced from root tissue known to be infected with the fungus as verified by microscopic examination. A similar PCR product was obtained from soil samples collected from fields with an established infection of P. sclerotioides on alfalfa. This PCR-based assay enables detection of P. sclerotioides from alfalfa root tissue and in soil samples in a single day, including extraction of DNA, compared with standard methods that require up to 100 days for identification using agar media.

Ethical issues in psychiatry and occupational medicine.

For the clinician involved in evaluation and treatment of the employee alleging work-related psychiatric dysfunction, the ethical challenges are many. The chapter provides a framework concerning the issues of ethics in medical/legal practice for the clinician working in occupational medicine and psychiatry.