Evaluation of Winter Ticks (Dermacentor albipictus) Collected from North American Elk (Cervus canadensis) in an Area of Chronic Wasting Disease Endemicity for Evidence of PrPCWD Amplification Using Real-Time Quaking-Induced Conversion Assay.

Chronic wasting disease (CWD) is a progressive and fatal spongiform encephalopathy of deer and elk species, caused by a misfolded variant of the normal prion protein. Horizontal transmission of the misfolded CWD prion between animals is thought to occur through shedding in saliva and other forms of excreta. The role of blood in CWD transmission is less clear, though infectivity has been demonstrated in various blood fractions. Blood-feeding insects, including ticks, are known vectors for a range of bacterial and viral infections in animals and humans, though to date, there has been no evidence for their involvement in prion disease transmission. In the present study, we evaluated winter ticks (Dermacentor albipictus) collected from 136 North American elk (Cervus canadensis) in an area where CWD is endemic for evidence of CWD prion amplification using the real-time quaking-induced conversion assay (RT-QuIC). Although 30 elk were found to be CWD positive (22%) postmortem, amplifiable prions were found in just a single tick collected from an elk in advanced stages of CWD infection, with some evidence for prions in ticks collected from elk in mid-stage infection. These findings suggest that further investigation of ticks as reservoirs for prion disease may be warranted. IMPORTANCE This study reports the first finding of detectable levels of prions linked to chronic wasting disease in a tick collected from a clinically infected elk. Using the real-time quaking-induced conversion assay (RT-QuIC), "suspect" samples were also identified; these suspect ticks were more likely to have been collected from CWD-positive elk, though suspect amplification was also observed in ticks collected from CWD-negative elk. Observed levels were at the lower end of our detection limits, though our findings suggest that additional research evaluating ticks collected from animals in late-stage disease may be warranted to further evaluate the role of ticks as potential vectors of chronic wasting disease.

Management of chronic wasting disease in ranched elk: conclusions from a longitudinal three-year study.

Chronic wasting disease is a fatal, horizontally transmissible prion disease of cervid species that has been reported in free-ranging and farmed animals in North America, Scandinavia, and Korea. Like other prion diseases, CWD susceptibility is partly dependent on the sequence of the prion protein encoded by the host's PRNP gene; it is unknown if variations in PRNP have any meaningful effects on other aspects of health. Conventional diagnosis of CWD relies on ELISA or IHC testing of samples collected post-mortem, with recent efforts focused on antemortem testing approaches. We report on the conclusions of a study evaluating the role of antemortem testing of rectal biopsies collected from over 570 elk in a privately managed herd, and the results of both an amplification assay (RT-QuIC) and conventional IHC among animals with a several PRNP genotypes. Links between PRNP genotype and potential markers of evolutionary fitness, including pregnancy rates, body condition, and annual return rates were also examined. We found that the RT-QuIC assay identified significantly more CWD positive animals than conventional IHC across the course of the study, and was less affected by factors known to influence IHC sensitivity - including follicle count and PRNP genotype. We also found that several evolutionary markers of fitness were not adversely correlated with specific PRNP genotypes. While the financial burden of the disease in this herd was ultimately unsustainable for the herd owners, our scientific findings and the hurdles encountered will assist future CWD management strategies in both wild and farmed elk and deer.

Cross-validation of the RT-QuIC assay for the antemortem detection of chronic wasting disease in elk.

Chronic wasting disease is a progressively fatal, horizontally transmissible prion disease affecting several members of the cervid species. Conventional diagnosis relies on ELISA or IHC evaluation using tissues collected post-mortem; however, recent research has focused on newly developed amplification techniques using samples collected antemortem. The present study sought to cross-validate the real-time quaking-induced conversion assay (RT-QuIC) evaluation of rectal biopsies collected from an elk herd with endemic CWD, assessing both binary positive/negative test results as well as relative rates of amplification between laboratories. We found that results were correlative in both categories across all laboratories performing RT-QuIC, as well as to conventional IHC performed at a national reference laboratory. A significantly higher number of positive samples were identified using RT-QuIC, with results seemingly unhindered by low follicle counts. These findings support the continued development and implementation of amplification assays in the diagnosis of prion diseases of veterinary importance, targeting not just antemortem sampling strategies, but post-mortem testing approaches as well.

First Report of Bacterial Leaf Spot of Swiss Chard Caused by Pseudomonas syringae pv. aptata in California.

From 1999 through 2003, a previously unreported disease was found on commercial Swiss chard (Beta vulgaris subsp. cicla) in the Salinas Valley, (Monterey County) California. Each year the disease occurred sporadically throughout the long growing season from April through September. Initial symptoms were water-soaked leaf spots that measured 2 to 3 mm in diameter. As disease developed, spots became circular to ellipsoid, 3 to 8 mm in diameter, and tan with distinct brown-to-black borders. Spots were visible from the adaxial and abaxial sides. Cream-colored bacterial colonies were consistently isolated from spots. Strains were fluorescent on King's medium B, levan positive, oxidase negative, and arginine dihydrolase negative. Strains did not rot potato slices but induced a hypersensitive reaction on tobacco (Nicotiana tabacum cv. Turk). The isolates, therefore, belong in LOPAT group 1 (1). Fatty acid methyl esters (FAME) analysis (MIS-TSBA version 4.10, MIDI Inc., Newark, DE) gave variable results that included Pseudomonas syringae, P. cichorii, and P. viridiflava with similarity indices ranging from 0.91 to 0.95. BOX-polymerase chain reaction (PCR) analysis gave identical banding patterns for the chard isolates and for known P. syringae pv. aptata strains, including the pathotype strain CFBP1617 (2). The bacteria were identified as P. syringae. Pathogenicity of 11 strains was tested by growing inoculum in nutrient broth shake cultures for 48 h, diluting to 10 × 6 CFU/ml, and spraying onto 5-week-old plants of Swiss chard cvs. Red, White, Silverado, and CXS2547. Untreated control plants were sprayed with sterile nutrient broth. After 7 to 10 days in a greenhouse (24 to 26°C), leaf spots similar to those observed in the field developed on all inoculated plants. Strains were reisolated from the spots and identified as P. syringae. Control plants remained symptomless. To investigate the host range of this pathogen, the same procedures were used to inoculate three strains onto other Chenopodiaceae plants: five cultivars of sugar beet (B. vulgaris), and one cultivar each of spinach (Spinacia oleracea) and Swiss chard. In addition, five chard strains and strain CFBP1617 were inoculated onto two cultivars of sunflower (Helianthus annuus), and one cultivar each of cantaloupe (Cucumis melo), sugar beet, spinach, and Swiss chard. All Swiss chard, cantaloupe, sunflower, and sugar beet plants developed leaf spots after 7 days. The pathogen was reisolated from spots and confirmed to be the same bacterium using BOX-PCR analysis. Spinach and untreated controls failed to show symptoms. All inoculation experiments were done at least twice and the results were the same. The phenotypic data, fatty acid and genetic analyses, and pathogenicity tests indicated that these strains are P. syringae pv. aptata. To our knowledge this is the first report of bacterial leaf spot of commercially grown Swiss chard in California caused by P. syringae pv. aptata. The disease was particularly damaging when it developed in Swiss chard fields planted for "baby leaf" fresh market products. Such crops are placed on 2-m wide beds, planted with high seed densities, and are sprinkler irrigated. This disease has been reported from Asia, Australia, Europe, and other U.S. states. References: (1) R. A. Lelliott et al. J. Appl. Bacteriol. 29:470, 1966. (2) J. L. W. Rademaker et al. Mol. Microbiol. Ecol. Man. 3.4.3:1-27, 1998.

Cursoriality in bipedal archosaurs.

Modern birds have markedly foreshortened tails and their body mass is centred anteriorly, near the wings. To provide stability during powered flight, the avian centre of mass is far from the pelvis, which poses potential balance problems for cursorial birds. To compensate, avians adapted to running maintain the femur subhorizontally, with its distal end situated anteriorly, close to the animal's centre of mass; stride generation stems largely from parasagittal rotation of the lower leg about the knee joint. In contrast, bipedal dinosaurs had a centre of mass near the hip joint and rotated the entire hindlimb during stride generation. Here we show that these contrasting styles of cursoriality are tightly linked to longer relative total hindlimb length in cursorial birds than in bipedal dinosaurs. Surprisingly, Caudipteryx, described as a theropod dinosaur, possessed an anterior centre of mass and hindlimb proportions resembling those of cursorial birds. Accordingly, Caudipteryx probably used a running mechanism more similar to that of modern cursorial birds than to that of all other bipedal dinosaurs. These observations provide valuable clues about cursoriality in Caudipteryx, but may also have implications for interpreting the locomotory status of its ancestors.

Outbreak of Leaf Spot of Saponaria Caused by Alternaria saponariae in California.

Saponaria (Saponaria vaccaria [= Vaccaria hispanica]) is a Caryophyllaceae plant that is grown commercially in California as a cut flower. In 1998, a leaf spot disease devastated the commercially grown saponaria in coastal California. The entire saponaria crop was completely unmarketable because of extensive leaf spotting. Symptoms consisted of circular, brown, necrotic leaf spots with diameters up to 8 mm and concentric zones of lighter and darker tissue. Chlorotic borders developed around the spots. Conidia from leaves were obclavate, usually had 7 transverse and 1 to 4 longitudinal septa, and narrowed gradually toward the apex into a blunt-tipped, unbranched beak cell. The spore body measured 69 to 90 (to 119) × 17 to 21 (to 25) µm, with the distinctive beak cell 17 to 53 µm long. Conidia formed short chains on host tissue. The fungus was identified as Alternaria saponariae (Peck) Neergaard (2). For pathogenicity tests, six representative isolates were grown on V8 juice agar under fluorescent tube lighting. Potted saponaria were sprayed with either conidial concentrations (1 × 10e5 conidia per ml) or water. Plants were incubated in a chamber with a humidifier for 48 h and then maintained in a greenhouse (23 to 25°C). After 14 days, leaf spots similar to the original symptoms developed on all inoculated plants, and the pathogen was reisolated. Plants sprayed with water were symptomless. The experiment was repeated and the results were similar. Using the same isolates and method, we inoculated carnation (Dianthus caryophyllus), sweet William (Dianthus barbatus), and saponaria. However, disease developed only on saponaria. While A. saponariae on saponaria was reported previously in California (1), this is the first report to characterize the pathogen and document that isolates are pathogenic on saponaria but not on other commercial Caryophyllaceae hosts. References: (1) K. F. Baker and L. H. Davis. Plant Dis. Rep. 34:403, 1950. (2) P. Neergaard. Aarsberet. J. E. Ohlsens Enkes Plantepat. Lab. No. 3, 1938.

A New Root and Crown Rot Disease of Heath in California Caused by Cylindrocladium pauciramosum.

Heath (Erica capensis Salter) is a woody, evergreen plant used in Cali-fornia as a landscape shrub or ground cover. In 1997, a new root and crown disease was found in commercial nursery plantings of potted heath. A similar disease was found in 1998 on heath transplants being grown as liners. In both situations, roots were necrotic and crown tissue turned brown. Affected plants became gray-green in color, withered, and died. A Cylindrocladium species was consistently isolated from roots, crowns, and lower stems of symptomatic plants. Isolates were characterized by having penicillate conidiophores terminating in obpyriform to broadly ellipsoidal vesicles. Conidia were hyaline, 1-septate, straight with rounded ends, (30-) 45 to 55 (-60) × (3.5-) 4 to 5 µm, placing it in the Cylindrocladium candelabrum Viégas species complex. Ten single-conidial isolates produced perithecia with viable progeny of Calonectria pauciramosa C.L. Schoch & Crous when mated on carnation leaf agar with tester strains of Cylindrocladium pauciramosum C.L. Schoch & Crous (1). Matings with tester strains of all other species in this complex proved unsuccessful. Pathogenicity of 8 representative isolates was confirmed by applying 3 ml of a conidial suspension (3.0 × 105 conidia per ml) to the crowns of potted, 6-month-old, rooted heath cuttings that were subsequently maintained in a greenhouse (23 to 25°C). After 2 weeks, plant crowns and roots developed symptoms similar to those observed in the field, and plants later wilted and died. C. pauciramosum was reiso-lated from all plants. Control plants, which were treated with water, did not develop any symptoms. The tests were repeated and the results were similar. This is the first report of C. pauciramosum as a pathogen of heath, and the first record of this pathogen from North America. Reference: (1) C. L. Schoch et al. Mycologia 91:286, 1999.

Black Root Rot, Caused by Thielaviopsis basicola, on Tomato Transplants in California.

In 1997, greenhouse-produced transplants of tomato (Lycopersicon esculentum) exhibited stunting, yellowing of leaves, and lack of vigorous growth. Roots of affected plants had numerous small (2 to 10 mm long), brown lesions. Isolations from symptomatic roots onto acidified potato dextrose agar and TBM-V8 medium (1) consistently resulted in gray fungal colonies that formed catenulate, cylindrical, hyaline endoconidia and catenulate, subrectangular, thick-walled, dark aleuriospores. The fungus was identified as Thielaviopsis basicola (Berk. & Broome) Ferraris based on colony characteristics and conidial morphology. Pathogenicity was tested by producing endoconidial suspensions of representative isolates and applying them as root drenches to 2-month-old tomato (cv. Early Girl) plants in soil-less, peat-based potting mix. Sterile, distilled water was applied to control plants. After 14 days in a greenhouse, symptoms similar to those originally observed developed and the pathogen was reisolated from lesions on the roots. Control plants developed no disease symptoms. The test was repeated and the results were similar. This is the first documentation of black root rot caused by T. basicola on tomato transplants in California. Disease incidence reached as high as 50 to 60% in certain plantings. Reference: (1) J. N. C. Maduewesi et al. Phytopathology 66:526, 1976.

First Report of Bell Pepper (Capsicum annuum) as a Host of Sclerotinia minor in California.

In 1997, commercially grown bell pepper in the Salinas Valley (Monterey County), California, developed a previously undescribed disease. Plant foliage became pale green and wilted. Crowns developed brown lesions that girdled the plants, resulting in plant death. White mycelia and small (2 to 3 mm), black, irregularly shaped sclerotia were observed on the outside of plant crowns and in the centers of stem cavities. Isolations consistently resulted in the recovery of Sclerotinia minor. Pathogenicity was tested by inoculating 2-month-old bell pepper plants (cv. California Wonder) with sclerotia from three pepper and three lettuce (Lactuca sativa) isolates of S. minor (seven plants per isolate). Six to 10 sclerotia were placed 1 cm below the soil line and adjacent to the plant crowns. After 8 days, plants inoculated with pepper and lettuce isolates developed symptoms similar to those found in commercial fields, and S. minor was recovered from all peppers. The uninoculated control plants developed no symptoms. This is the first report of bell pepper as a host of S. minor.

Phytophthora Root and Crown Rot of Sage Caused by Phytophthora cryptogea in California.

In 1996, commercial plantings of sage (Salvia officinalis) in the Salinas Valley in Monterey County, CA, were affected by a root and crown disease. Roots were necrotic, and crowns and lower stems turned black. Affected plants withered and died. A Phytophthora sp. was consistently isolated from roots and stems of the symptomatic plants. The species was identified as Phytophthora cryptogea based upon the formation of hyphal swellings, morphology of sporangia and oospores, and growth at cardinal temperatures (1). Pathogenicity of representative isolates was confirmed by applying 2 ml of a zoospore suspension (2.0 × 105 zoospores per ml) to roots and crowns of 3-month-old potted sage plants. After treatment, plants were placed for 24 h in shallow trays of water to saturate the root zone, then were removed from trays and incubated in a greenhouse. After 4 days, foliage of all inoculated plants began to wilt. After 7 days, plant crowns and stems turned black and the plants collapsed. P. cryptogea was reisolated and recharacterized from all plants. Control plants, which were treated with water and then handled in the same way as inoculated plants, did not develop any symptoms. The tests were repeated and the results were similar. This is the first report of P. cryptogea attacking commercial plantings of sage. The authors also detected this disease in experimental plantings of sage in Stanislaus County in 1990. Reference: (1) D. C. Erwin and O. K. Ribeiro. 1996. Phytophthora Diseases Worldwide. American Phytopathological Society, St. Paul, MN.

Molecular characterization of the ldmdr1 multidrug resistance gene from Leishmania donovani.

The ldmdr1 gene that confers resistance to multiple structurally dissimilar hydrophobic drugs in Leishmania donovani has been isolated within a 5.4-kb XmaI fragment from a genomic library of L. donovani DNA and its protein coding region sequenced. The longest open reading frame within ldmdr1 encodes a 146.5-kDa protein of 1341 amino acids, designated LDMDR1. The primary structure and predicted membrane topology of LDMDR1 indicates that it is a member of the P-glycoprotein superfamily with the greatest homology to the mammalian multidrug resistance P-glycoproteins. A 2.3-kb SalI fragment derived from a second ldmdr1 allele was also cloned from the L. donovani library. Nucleotide sequence analysis of a portion of the SalI insert revealed 5 single base differences from its counterpart within the 5.4-kb XmaI fragment, one of which created a PvuI restriction site polymorphism. Southern blots of PvuI-digested DNA divulged that the amplified ldmdr1 gene copies in a multidrug-resistant L. donovani strain were all derived from the single ldmdr1 allele whose protein coding segment was sequenced in its entirety.

Cloning of the gene encoding Leishmania donovani S-adenosylhomocysteine hydrolase, a potential target for antiparasitic chemotherapy.

A full-length gene encoding the S-adenosylhomocysteine hydrolase (AdoHcyase) enzyme has been isolated from a genomic library of Leishmania donovani DNA in lambda GEM-11 by cross-hybridization to the full-length human AdoHcyase cDNA. The nucleotide sequence of the SalI fragment contained a single open reading frame that encoded a polypeptide of 438 amino acids (47,712 Da). After maximum gap alignment, the predicted amino acid sequence of the leishmanial AdoHcyase was 70-73% identical to AdoHCyases from higher eukaryotes. In addition, a data base search revealed that the primary structure of all AdoHcyase proteins was highly homologous to that of a protein encoded by a mRNA from Drosophila melanogaster that maps near the r element function of the Abd-b homeotic gene. In Northern blots, the SalI fragment hybridized to a 3.0-kb transcript that presumably encodes the parasite enzyme. Southern blot analysis of genomic DNA revealed that the AdoHcyase gene did not exist as a tandemly repeated array within the L. donovani genome. Moreover, monoclonal antibodies generated against human AdoHcyase recognized a leishmanial protein on immunoblots. Finally, the growth of L. donovani promastigotes could be arrested by micromolar concentrations of 3-deazaaristeromycin (C3Ari) and 9-(trans-2',trans-3'-dihydroxycyclopentanyl)adenine, 2 known inhibitors of mammalian AdoHcyase. C3Ari also induced a substantial expansion of the intracellular pools of both AdoHcy and S-adenosylmethionine (AdoMet), as well as a significant diminution of the AdoMet/AdoHcy ratio. Thus, AdoHcyase may have therapeutic potential for the selective treatment of diseases of parasitic origin.

Multidrug resistance in Leishmania donovani is conferred by amplification of a gene homologous to the mammalian mdr1 gene.

Drug resistance is a major impediment to the effective treatment of parasitic diseases. The role of multidrug resistance (mdr) genes and their products in this drug resistance phenomenon, however, remains controversial. In order to determine whether mdr gene amplification and overexpression can be connected to a multidrug resistance phenotype in parasitic protozoa, a mutant strain of Leishmania donovani was generated by virtue of its ability to proliferate in medium containing increasing concentrations of vinblastine. The vinblastine-resistant strain, VINB1000, displayed a cross-resistance to puromycin and the anthracyclines, a growth phenotype that could be attributed to an impaired ability to accumulate the toxic drugs. By using the polymerase chain reaction, two different DNA fragments, LEMDR06 and LEMDRF2, were amplified from leishmanial genomic DNA, and each amplified fragment encoded a product that was significantly homologous to parts of the mammalian P-glycoprotein. In the VINB1000 strain, the mdr gene recognized by the LEMDR06 probe was amplified approximately 50-fold in copy number, whereas the mdr genes that hybridized to LEMDRF2 or to a fragment of the previously characterized ltpgpA gene were not amplified. Moreover, the VINB1000 cell line expressed a LEMDR06 gene transcript of 12.5 kb in size that was not detected in the parental wild-type strain. To furnish a functional test for mdr gene amplification and expression in L. donovani, the L. donovani gene recognized by the LEMDR06 polymerase chain reaction product, ldmdr1, was isolated from a genomic library, transfected into wild-type cells, and amplified over 500-fold by selection in 0.5 mg of G418 per ml. The resulting transfectants were resistant to all drugs to which VINB1000 cells were resistant and sensitive to all drugs to which VINB1000 cells were sensitive. These studies demonstrate that amplification of the ldmdr1 gene either by direct selection or subsequent to transfection can confer a drug-resistant phenotype in parasitic protozoa similar to that observed for MDR mammalian cells.

Pulse compression of an FM chirped CO(2) laser.

A CO(2) laser has been FM chirp modulated by a CdTe intracavity modulator. A frequency deviation-of-100 MHz in 2 micros was attained in this fashion. Following heterodyne detection the chirped pulse was compressed to 15 ns using a surface acoustic wave compression filter. This corresponded to a compression factor of 130. The suppression of unwanted sidelobes with a weighting filter was demonstrated. We have explored the use of this technique for laser radar systems and described an electrooptically FM modulated CO(2) waveguide laser with postdetection pulse compression by a surface acoustic wave compressive filter. To our knowledge this is the first report of the successful operation of this important system.

Waveguide lasers with intracavity electrooptic modulators: misalignment loss.

Coupling from the EH(11) fundamental mode of a square, hollow bore waveguide laser to a square electrooptic modulator is treated. The misalignment loss that results from coupling of the laser fundamental mode into higher order modulator modes is calculated. Results are presented for loss from transverse displacement, angular tilt, and axial separation of the two elements. From these results, the alignment tolerances required to minimize loss for an intracavity modulator are found.