Multiplex Polymerase Chain Reaction Identification of Single Individuals of the Longidorid Nematodes Xiphinema index, X. diversicaudatum, X. vuittenezi, and X. italiae Using Specific Primers from Ribosomal Genes

ABSTRACT The species X. index, X. diversicaudatum, X. vuittenezi, and X. italiae are established (E) or putative (P) vectors of Grapevine fanleaf virus (GFLV) (E), Arabis mosaic virus (E), Grapevine chrome mosaic virus (P), and GFLV (P) nepoviruses of grapevine, respectively. All four species are very closely related taxonomically and their low field densities make them difficult to identify from morphological and morphometrical diagnostic characters when only single or few individuals are detected. To improve diagnostic accuracy, a simple method was developed. The internal transcribed spacer 1 (ITS1) region spanning the 18S and 5.8S ribosomal genes was sequenced in one population of each species using two conserved primers from these genes. The ITS1 fragments were 1,132 bp (X. vuittenezi), 1,153 bp (X. index), 1,175 bp (X. diversicaudatum), and 1,190 bp (X. italiae), i.e., a difference of over 5% between the extremes. The sequence variability made it possible to design species-specific internal sense primers that amplified, in combination with the same antisense ITS1 primer, a single signature fragment (340 bp for X. index, 414 bp for X. italiae, 591 bp for X. vuittenezi, and 813 bp for X. diversicaudatum). Tests with DNA from a single adult or juvenile nematode confirmed the specificity of the primers from diverse isolates or populations. The primers were successfully used in a multiplex test for the reliable detection of two to four mixed species, each represented by a single individual. This multiplex-based diagnostic tool will be particularly useful for successful nematode management practices in vineyards.     

Diagnosis of three <Emphasis Type="Italic">Tospovirus</Emphasis> species by rapid immunofilter paper assay

The rapid immunofilter paper assay (RIPA) was developed to detect Tomato spotted wilt virus (TSWV), Groundnut ringspot virus (GRSV), and Tomato chlorotic spot virus (TCSV) using antisera against recombinant nucleocapsid (N) proteins of each tospovirus. The two-step RIPA was sensitive enough to detect each pecies specifically in only 30min. This technique is proposed as an excellent tool for routine Tospovirus diagnosis and field epidemiological studies.     

United States Department of Agriculture-Agricultural Research Service research on alternatives to methyl bromide: pre-plant and post-harvest

Methyl bromide is a widely used fumigant for both pre-plant and post-harvest pest and pathogen control. The Montreal Protocol and the US Clean Air Act mandate a phase-out of the import and manufacture of methyl bromide, beginning in 2001 and culminating with a complete ban, except for quarantine and certain pre-shipment uses and exempted critical uses, in January 2005. In 1995, ARS built on its existing programs in soil-borne plant pathology and post-harvest entomology and plant pathology to initiate a national research program to develop alternatives to methyl bromide. The focus has been on strawberry, pepper, tomato, perennial and nursery cropping systems for pre-plant methyl bromide use and fresh and durable commodities for post-harvest use. Recently the program has been expanded to include research on alternatives for the ornamental and cut flower cropping systems. An overview of the national research program is presented. Results from four specific research trials are presented, ranging from organic to conventional systems. Good progress on short-term alternatives is being made. These will be used as the foundation of integrated management systems which begin with pre-plant management decisions and continue through post-harvest processing.     

An age-induced switch to a hyper-recombinational state

There is a strong correlation between age and cancer, but the mechanism by which this phenomenon occurs is unclear. We chose Saccharomyces cerevisiae to examine one of the hallmarks of cancer--genomic instability--as a function of cellular age. As diploid yeast mother cells aged, an approximately 100-fold increase in loss of heterozygosity (LOH) occurred. Extending life-span altered neither the onset nor the frequency of age-induced LOH; the switch to hyper-LOH appears to be on its own clock. In young cells, LOH occurs by reciprocal recombination, whereas LOH in old cells was nonreciprocal, occurring predominantly in the old mother's progeny. Thus, nuclear genomes may be inherently unstable with age.     

Pathogenesis of chicken-passaged Newcastle disease viruses isolated from chickens and wild and exotic birds

The pathogenesis of six Newcastle disease virus (NDV) isolates recovered from chickens (Ckn-LBM and Ckn-Australia) and wild (Anhinga) and exotic (YN parrot, pheasant, and dove) birds was examined after the isolates had been passaged four times in domestic chickens. Groups of 10 4-wk-old specific-pathogen-free white leghorn chickens were inoculated intraconjunctivally with each one of the isolates. The infected birds were observed for clinical disease and were euthanatized and sampled at selected times from 12 hr to 14 days postinoculation or at death. Tissues were examined by histopathology, by immunohistochemistry (IHC) to detect viral nucleoprotein (IHC/NP), and by in situ hybridization to detect viral mRNA and were double labeled for apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling ([TUNEL] or IHC/caspase-3) and viral nucleoprorein (IHC/NP). Birds infected with the three low virulence viruses (Ckn-LBM, YN parrot, and Ckn-Australia) did not develop clinical disease. Microscopic lesions were observed only at the inoculation site and in organs of the respiratory system. The detection of viral nucleoprotein (N) was restricted to the inoculation site. The pheasant and dove isolates were highly virulent for chickens with marked tropism for lymphoid tissues, confirmed by the presence of large numbers of cells positive for viral N protein and viral mRNA. Viral N protein was detected early in the cytoplasm of cells in the center of the splenic ellipsoids. The apoptosis assays (TUNEL and IHC/caspase-3) showed increased apoptosis in the splenic ellipsoids as well. Apparently, apoptosis is an important mechanism in lymphoid depletion during NDV infection.     

Ionophores have limited effects on jejunal glucose absorption and energy metabolism in mice

Two experiments, Trial 1 (in vitro) and Trial 2 (in vivo), were conducted to examine the effects of ionophores, monensin, laidlomycin, and laidlomycin propionate on whole-animal O2 consumption, organ weights, jejunal glucose absorption, and O2 utilization, as well as growth, feed and water consumption, and feed efficiency. In Trial 1, 30 male Swiss-Webster mice, 8 wk old, were used to measure the in vitro effects of each of the ionophores at concentrations of 1.62 or 16.2 mM. Six combinations of three ionophores at two concentrations resulted in a total of eight treatments. All eight treatments were exposed to jejunal rings from a single mouse for a total of 30 observations per treatment. Jejunal rings were exposed to each ionophore treatment for 15 min. Laidlomycin propionate (16.2 mM) decreased (P < 0.02) glucose absorption, as estimated by H3-3-O-methyl glucose uptake compared with all other treatments, whereas laidlomycin propionate (1.62 mM) increased (P = 0.032) jejunal DM content compared with 16.2 mM laidlomycin propionate. In Trial 2, 40 5-wk-old mice were allotted into four treatments--control and 16.2 mM each of monensin, laidlomycin, and laidlomycin propionate--for a total of 10 observations per treatment. Ionophores were administered via the drinking water for 14 d. No ionophore treatment had any effect on whole-mouse O2 consumption. Monensin increased (P = 0.004) stomach size and decreased (P = 0.049) the efficiency of BW gain compared with controls. Laidlomycin propionate decreased (P = 0.032) the percentage of whole jejunum oxygen consumption due to oubain-sensitive respiration compared with control. The efficiency of intestinal glucose absorption was not changed due to treatment in either trial. Under the conditions of these studies, monensin, laidlomycin, and laidlomycin propionate had minimal and inconsistent effects on jejunal function and energy utilization in mice. This investigation suggests that changes in the energetic requirements of animals treated with ionophores are not an issue in animal production.     

Evaluation of frequent milkout for treatment of cows with experimentally induced Escherichia coil mastitis

OBJECTIVE: To evaluate the effect of frequent milkout (FMO) on the outcome of experimentally induced Escherichia coli mastitis in cows. DESIGN: Randomized complete block study. ANIMALS: 16 Holstein dairy cows. PROCEDURE: Cows were randomly assigned to 1 of 4 groups and were either not infected and not treated (NI-NT), experimentally infected with E coli and not treated (EC-NT), not infected and FMO (NI-FMO), or experimentally infected with E coli and FMO (EC-FMO). The infected quarter in cows in FMO groups was milked out every 4 hours from 16 to 36 hours and every 6 hours from 36 to 84 hours after challenge, with the aid of oxytocin administration. Somatic cell counts (SCC); times to bacterial, clinical, and systemic cures; and serum concentrations of a-lactalbumin were determined. RESULTS: Use of FMO did not appear to affect SCC. For EC-NT and EC-FMO groups, mean bacterial cure times were 203 and 159 hours, clinical cure times were 276 and 360 hours, and systemic cure times were 144 and 159 hours, respectively; these times were not significantly different. Concentrations of a-lactalbumin were significantly increased in the EC-NT group at 12 hours and in the NI-FMO group at 36 and 60 hours after challenge, compared with values of cows in other treatment groups. CONCLUSIONS AND CLINICAL RELEVANCE: Compared with results in control cows, FMO does not appear to be an efficacious treatment for experimentally induced moderate to severe E coli mastitis.     

Evaluation of relationships between pretreatment patient variables and duration of isolation for radioiodine-treated hyperthyroid cats

OBJECTIVE: To determine relationships between commonly measured pretreatment variables and duration of isolation for unrestricted dismissal after oral administration of iodine 131 (131I) for treatment of hyperthyroidism in cats. ANIMALS: 149 hyperthyroid cats treated with 131I. PROCEDURE: A dose of 131I (2.9 to 6.04 mCi [1.07 to 2.23 x 10(8) Bq]) was administered orally to all cats after hyperthyroidism was confirmed by evaluation of serum total thyroxine (T4) concentrations. Forward stepwise regression analysis was used to determine whether pretreatment total T4 concentration, serum creatinine concentration, body weight, age, 131I dose, or concurrent administration of cardiac medication (specifically excluding thyroid suppression drugs) could be used as pretreatment predictors of duration of isolation in a clinical setting. Gamma radiation emission rate at dismissal was < 2.0 mR/h at skin surface over the thyroid region. RESULTS: Mean +/- SD duration of isolation was 16.67 +/- 4.42 days (95% confidence interval, 9.2 to 24.1 days). The regression equation for duration of isolation calculated on the basis of dose of 131I (duration of isolation [days] = 3.2 + [2.66 X mCi - 131I dose]) yielded a regression line with a 95% confidence interval of +/- 3.3 days; only 15% of the variation was explained. CONCLUSIONS AND CLINICAL RELEVANCE: A pretreatment estimate for duration of isolation could be determined only from an equation based on the orally administered dose of 131I. These findings suggest that administration of the lowest efficacious dose possible is the dominant factor in reduction of duration of isolation for cats treated with 131I.