Genotypic and Phenotypic Characterization of Streptomyces Species Causing Potato Common Scab in Uruguay.

Isolation and characterization of common scab (CS) pathogen Streptomyces spp. from Uruguayan potato tubers and soil samples were done in response to significant economic losses due to CS on potato in autumn 2010. Seventy of the 331 isolates were classified as pathogenic owing to their ability to induce necrosis on tuber disks and stunting of radish seedling. Streptomyces spp. causing CS on potato in Uruguay were found to represent a range of different species by virtue of their diverse morphological and physiological traits as well as rep-PCR, rpoB phylogenetic analysis, and multi-locus sequences analysis. We identified isolates primarily as Streptomyces scabiei, S. acidiscabies, and S. europaeiscabiei. However, some of the pathogenic isolates still remain to be identified at the species level. This highlights the need for improved methods for discrimination among pathogenic Streptomyces species. The presence of Streptomyces pathogenicity island (PAI) genes was analyzed, including genes encoding for thaxtomin synthetase (txtA, txtB), tomatinase (tomA), and a necrosis protein (nec1). Among the isolates that were pathogenic, 50% contained the four pathogenicity genes, 33% had an atypical composition of PAI marker genes, and 17% did not contain any genes. The absence of the genes reported to be involved in thaxtomin biosynthesis (txtA, txtB) was confirmed by whole-genome sequencing of two representative strains of this group. This finding suggests the participation of other virulence factors in plant pathogenicity.

Susceptibility of mice to group B coxsackie virus is influenced by the diabetic gene.

A positive correlation was found between genetic predisposition to diabetes in the mouse and susceptibility to group B Coxsackie virus in this host. Male mice of the inbred strain C57BL/Ks and the following genetic variants were used; mice homozygous for the autosomal recessive gene for diabetes (db/db), the phenotypically normal heterozygous (db/+), and the normal mice which lacked the diabetic gene (+/+). The mortality response of the +/+ mice to intraperitoneal inoculation with Coxsackie virus B4 differed from the response of the two genetic variants (db/db and db/+) derived from this strain. The db/+ variant was more susceptible to Coxsackie virus B4 than the parental background strain (+/+). The db/db variant was more susceptible than either of the other genotypes. Pathological findings of the pancreas of the three genotypes during the acute stage of infection closely paralleled the genotypically dependent susceptibility of the host.

Immunodeficiency as primary phenotype of diabetes mutation db. Studies with coxsackievirus B4.

Restriction of food intake (R) in the C57BL/KsJ db/db diabetic mutant mouse prevents phenotypic expression of diabetes, whereas ad libitum feeding (AL) results in spontaneous diabetes. Previous results showed that coxsackievirus B4 (CB4)-infected genetically identical db/db mice with and without diabetes could be distinguished by the levels of CB4-neutralizing antibody and virus-specific antibodies as determined by enzyme-linked immunosorbent assay and the numbers of splenic antibody-forming cells. Our results show that the diabetic genotype db/db R was deficient in total spleen lymphocytes and lymphocyte subsets and was unable to produce agglutinating antibody to sheep erythrocytes (SRBCs) or specific antibody to noninfectious CB4. The db/db AL mutant expressing the diabetic phenotype was not as deficient in spleen cell parameters. The response to noninfectious CB4 was delayed but substantial. The db/db AL mouse was also unique with its higher agglutinating antibody levels after virus infection than its uninfected control or the infected or uninfected db/db R mouse. In vitro SRBC immunization of spleen lymphocytes determined that this enhanced response was largely dependent on the diabetic milieu and was not a property of the cells. Genetic predisposition to diabetes is characterized by immunodeficiency as evident from inadequate levels of antibodies to infectious or noninfectious antigens and absolute and relative deficiency in spleen lymphocyte subsets and total numbers of spleen cells. Phenotypic expression of diabetes results in partial amelioration of the immunodeficiency evident in diabetic genotype db/db R without disease.

Platelet aggregation in the cerebral and mesenteric microcirculation of mice with genetically determined diabetes.

Platelet aggregates were induced in pial arterioles of the following strains of mice with a genetic predisposition for diabetes: ob/ob and db+/db+. Aggregation was compared with that in 6J+/+ mice, the nondiabetic controls for ob/ob animals, and in +m/+m as well as db+/+m, the nondiabetic controls for db+/db+ mice. Aggregation was also induced within mesenteric arterioles of db+/db+ animals and compared with that in db+/+m mice. Aggregation was monitored microscopically, by measuring the time required for a noxious stimulus to initiate aggregation in an injured arteriole. Platelet aggregation was initiated with equal ease in the pial arterioles of ob/ob mice and their 6J+/+ controls. However, the onset of aggregation in the pial arterioles of the db+/db+ group was significantly delayed when compared with the onset in either of the nondiabetic control groups, +m/+m or db+/+m. A similar prolongation in the time required to produce aggregation was also observed in the mesenteric arterioles of the db+/db+ mice when compared with db+/+m controls. The basis for reduced platelet aggregation in the microcirculation of db+/db+ mice is not explained. The results differ from those showing enhanced aggregation in many in vitro studies of platelets from human diabetics and from those of in vivo studies of two other animal models described in the literature. However, not all published studies have reported enhanced aggregation. The delayed aggregation in the present study may provide a basis for analysis of factors that regulate platelet aggregation in diabetes.

Contrasting effects of alpha- and beta-androstenediol on oncogenic myeloid cell lines in vitro.

The in vitro effects of 17 alpha AED, the isomer of 5-androstene-3 beta,17 beta diol (17 beta AED) on the basal growth of murine RAW 264.7, P388D1, and human HL-60 cells were investigated. 17 alpha AED treatment of RAW cells for 48 h reduced total cell number without increasing cell death as detected by trypan blue exclusion. At these doses, DNA synthesis as measured by [3H]thymidine incorporation was suppressed by as much as 65%, P < 0.05. This effect was time- and dose-dependent and reversible on removal of the steroid. Similar results were obtained with P388D1 and human HL-60 cell lines. At 50 nM or above, 17 alpha AED induced apoptosis in RAW cells and HL-60 as detected by transmission electron microscopy and TUNEL assays. By contrast, treating cells with the isomer 17 beta AED had no such effect. These data suggest that the balance between the anti-proliferative effect of 17 alpha AED and the proliferative effects of 17 beta AED may determine the overall level of myelopoiesis.

nec1, a gene conferring a necrogenic phenotype, is conserved in plant-pathogenic Streptomyces spp. and linked to a transposase pseudogene.

We are investigating the genetic basis for, and evolution of, plant pathogenicity in Streptomyces spp. The plant-pathogenic species S. scabies, S. acidiscabies, and S. turgidiscabies cause the scab disease of potato and produce the phytotoxins, thaxtomins. Forty-three Streptomyces strains representing the three species were evaluated; all thaxtomin A-producing Streptomyces strains were pathogenic on potato tubers and all but one hybridized to nec1 and ORFtnp, two genes previously cloned from S. scabies ATCC 41973. nec1 confers a pathogenic phenotype on S. lividans TK24, a nonpathogen, and ORFtnp is a transposase pseudogene located 5' to nec1. The eight nonpathogenic strains tested neither produced thaxtomin A nor hybridized to nec1. ORFtnp and nec1 occurred on a single PvuII restriction fragment in all thaxtomin A-producing Streptomyces strains. The nucleotide sequences of the homologs of nec1 and ORFtnp from two pathogenic strains each of S. scabies, S. acidiscabies, and S. turgidiscabies were identical; oligonucleotide primers specific to this gene amplified homologs from all strains that hybridized to nec1. We propose that nec1 and ORFtnp have been horizontally mobilized from S. scabies to S. acidiscabies and S. turgidiscabies, and that nec1 is involved in pathogenicity and physically linked to the thaxtomin A biosynthetic genes.

Pathophysiological aspects of coxsackievirus B intestinal infection.

Our findings reveal that intestinal infection with coxsackie B5 results in decreased intestinal epithelial cell division in association with an increase in carbohydrate (glucose) and amino acid (leucine) absorption in the small intestine. These findings are contrasted with those occurring during Salmonella infection, which results in increased intestinal cell division rate but decreased carbohydrate (glucose) absorption. The changes in intestinal function and physiology that have been described occurred during an asymptomatic viral infection characterized by normal intestinal histology. A reasonable hypothesis is that these pathophysiological changes may be due not only to a variety of local factors but also to hormonal effects induced by systemic spread of coxsackievirus B.

Coxsackievirus B cardiopathy and angiopathy in the hypercholesterolemic host.

Studies on the pathogenic potential of the human cardiotropic enterovirus, coxsackievirus B5, show that this agent localizes and replicates in the aorta of mice. Nutritionally-induced hypercholesterolemia leads to an increased replication and persistence of virus in tissues, specifically the aorta. Coxsackievirus B cardiopathy is markedly augmented in the hypercholesterolemic host, resulting in a persistent cardiomyolysis which is not evident in virus-infected animals with normal cholesterol levels. Pathological changes in the aorta become evident only months after the acute infection, and only in hypercholesterolemic animals previously infected with coxsackievirus B5. Our findings of coxsackievirus B-induced angiopathy and cardiopathy in the hypercholesterolemic host extend the known pathogenic range of these human viruses, and further emphasizes their potential as etiological agents of cardiovascular disease.

Endocrine regulation of murine macrophage function: effects of dehydroepiandrosterone, androstenediol, and androstenetriol.

In these studies, the in vitro influences of dehydroepiandrosterone (DHEA), androstenediol (AED), and androstenetriol (AET) on proinflammatory cytokine production from macrophages was examined. From physiologic to pharmacologic doses, DHEA suppressed secretion of each pro-inflammatory cytokine while AED had little influence on the responses. In sharp contrast, AET augmented TNF-alpha and IL-1 secretion while not influencing IL-6 production. Furthermore, the antiglucocorticoid activity of DHEA, AED, and AET was also investigated. Co-culture with AET counteracted the down-regulatory effect of hydrocortisone on LPS-induced TNF-alpha and IL-1 secretion. These data imply that AET is capable of regulating cytokine secretion from macrophages and may function to counterbalance glucocorticoid function.

Endocrine regulation of the immune response to influenza virus infection with a metabolite of DHEA-androstenediol.

In these studies the influence of androstenediol on the course of an experimental virus infection was examined. Pretreatment with 320 mg/kg AED protected male mice from lethal influenza virus infection. In addition, AED enhanced antigen-induced trafficking of mononuclear cells into the draining lymph node and augmented antigen-specific activation of helper-T cells, which are important for control of viral pathogenesis. Furthermore, AED prevented the characteristic increase in serum corticosterone noted during influenza A virus infection. Although steroid hormones, at least corticosteroids, typically suppress host immune and inflammatory responses in vivo, these data suggest that AED may function to augment host immune and inflammatory responses in contrast to corticosteroids.

Regulation of the immune response by dehydroepiandrosterone and its metabolites.

Dehydroepiandrosterone (5-androsten-3 beta-ol-17-one, DHEA) has been shown to protect mice from a variety of lethal infections. This includes, but is not limited to, infection with viruses (herpes virus type 2, coxsackie virus B4 (CB4)), bacteria (Enterococcus faecalis, Pseudomonas aeruginosa), and a parasite (Cryptosporidium parvum). We have previously reported that androstenediol (5-androstene-3 beta, 17 beta-diol, AED), derived from DHEA, is at least 100 x more effective in up-regulating systemic resistance against CB4 infection than its precursor. Furthermore, androstenetriol (5-androstene-3 beta,7 beta, 17 beta-triol, AET) which is formed by 7 beta hydroxylation of AED, was more effective against CB4 infection than its precursor, AED. Neither steroid, however, has shown any significant direct antiviral effects. The in vitro influences of DHEA, AED and AET on a mitogen-induced mixed splenocyte proliferation assay were determined. The results showed that DHEA suppressed the proliferation of concanavalin A (ConA)- or lipopolysaccharide-activated cultures in a dose-dependent manner. AED had little influence on the activation response. However, AET potentiated the response to both mitogens significantly above the control level. The regulation of interleukin (IL)-2 and IL-3 secretion from ConA-activated lymphocytes was analogous to these observations. These functions were depressed by DHEA, unaffected by AED, and potently increased by AET. Moreover, the classic immunosuppressive effects of hydrocortisone on ConA-induced lymphocyte proliferation, as well as IL-2 and IL-3 production, were unaffected by co-culture with DHEA and only minimally counteracted by AED. In contrast. AET significantly counteracted the effect of hydrocortisone when co-cultured together. These data show that while DHEA, AED and AET each function in a similar manner in vivo, in vitro their effects are dramatically different from one another with only AET potentiating the cellular response by increasing lymphocyte activation and counteracting the immunosuppressive activity of hydrocortisone.

Antiglucocorticoid function of androstenetriol.

The anti-inflammatory and immunosuppressive functions of corticosteroids have been well established and characterized. In contrast, a different group of native steroids, which include dehydroepiandrosterone (DHEA) and two of its metabolites, androstenediol (5-androstene-3 beta-17 beta-diol, AED) and androstenetriol (5-androstene-3 beta-7 beta-17 beta-triol, beta AET), function in vivo to up-regulate host immune response against infections and counteract stress-induced immunosuppression. Indeed, DHEA and particularly, AED and beta AET, have been shown to protect mice from viral, bacterial, and parasitic infections. In vivo, these three hormones are in opposition to the widely demonstrated immunosuppressive action of glucocorticoids, suggesting a possible new immune regulation mechanism. The individual activity in vitro of each of these steroids, i.e. DHEA, AED, and beta AET, on a mitogen-induced mixed splenocyte proliferation assay were determined. The results showed that DHEA suppressed the proliferation of cultures activated with concanavalin A (ConA) or lipopolysaccharide (LPS) in a dose-dependent manner. AED had little influence on the activation response. However, beta AET potentiated the response to both mitogens significantly above control. The regulation of the cytokine secretion, of both interleukin-2 (IL-2) and interleukin-3 (IL-3), from ConA-activated lymphocytes was affected in the same manner. These functions were depressed by DHEA, unaffected by AED, and potently increased by beta AET. Moreover, the classic immunosuppressive effects of hydrocortisone on ConA-induced lymphocyte proliferation, as well as on IL-2 and IL-3 production, were unaffected by being co-cultured with DHEA and only minimally counteracted by AED at high doses. In contrast, co-culturing with beta AET significantly counteracted the immunosuppressive effects of hydrocortisone on lymphocyte proliferation and cytokine production. These data show that in-vivo, DHEA, AED, and beta AET may have some similar functions, while in vitro, their effects are dramatically different from one another. Only beta AET could markedly potentiate the cellular response by increasing lymphocyte activation and counteracting the immnosuppressive activity of hydrocortisone on lymphocyte proliferation and cytokine production.

Mobilization of cutaneous immunity for systemic protection against infections.

This laboratory reported that a single subcutaneous (SC) injection of the natural steroid hormone dehydroepiandrosterone (DHEA) resulted in significant protection against a lethal herpes virus type 2 encephalitis or a systemic coxsackievirus B4 infection. Our previous results have shown that SC injection of DHEA resulted in upregulation of the specific host immune response resulting in protection against a lethal infection. This hormone did not have any direct antiviral effects in vitro. Furthermore, results indicate that, in vivo, DHEA is not the agent directly mediating the upregulation of the immune response. In the skin, DHEA is converted to androstenediol (AED) and it, in turn, is converted to androstenetriol; this is a metabolic process which appears unique to the skin. This report demonstrates that SC injection of AED results in markedly greater resistance against both viral and bacterial infection. Both DHEA and AED appear to function by facilitating and upregulating host immune responses via mobilization of cutaneous immunity to obtain systemic protection against infections. Because these steroids are native to the host and are regulated by the central nervous system, it is suggested that they may be an integral element of neuroimmunomodulation.

Steroid hormone regulation of antiviral immunity.

Recent observations in both humans and animals have demonstrated that stress is immunomodulatory and can alter the pathogenesis of microbial infections to the extent that it may be adverse to health. Stress disrupts homeostasis, and the body responds through endocrine and nervous system interactions in an effort to re-establish the health of the host. However, the resulting physiologic changes associated with stress, such as the rise in serum glucocorticoids (GCs), are implicated in suppression of antiviral immunity. Therefore, it would be of significance to counterregulate stress-mediated immunosuppression during viral infection to improve immune responses and limit virus-mediated damage. The data in this study focus upon the antiglucocorticoid influence of a native steroid hormone that has been shown to augment immune function and protect animals against lethal viral infections. Androstenediol (5-androstene-3 beta,17 beta-diol, AED), a metabolite of dehydroepiandrosterone (DHEA), confers protection against lethal infection with influenza A virus. The protective activity appears to counterbalance the function of the regulatory GCs because AED prevents GC-mediated suppression of IL-1, TNF-alpha, and IL-2 secretion. Furthermore, AED inhibits GC-induced transcription of a GC-sensitive reporter gene.

Androstenetriol and androstenediol. Protection against lethal radiation and restoration of immunity after radiation injury.

Androstenetriol (AET) and Androstenediol (AED) upregulate host immunity, leading to increased resistance against infections. AET augments IL-2, IL-3, IFN gamma levels, and counteracts hydrocortisone immune suppression. AET and AED at a dose of 0.75 mg/- and 8.0 mg/25-g mouse, protected 60 and 70%, respectively, of C57/BL/6J mice irradiated with a lethal dose. These hormones also protected mice irradiated with 6 Gy and infected with a coxsackievirus B4 LD50. AET significantly increased spleen lymphocyte numbers at 7, 14, and 21 days after a 6-Gy exposure. Fluorescent activated cell-sorter analysis of irradiated mice, spleen, and bone marrow showed that AET significantly augmented the myeloid precursor markers, CD11b/Mac-1, and B220 (pan B), as well as the absolute numbers of CD4+/CD8+ cells over the 21 days of testing. Overall, the data are consistent with AET/AED inducing a more rapid recovery of all hematopoietic precursors from the small number of surviving stem cells.

Androstenediol-induced restoration of responsiveness to influenza vaccination in mice.

Androstenediol (AED), a metabolite of dehydroepiandrosterone (DHEA) regulates innate and adaptive immune responses. To examine whether AED could effectively reverse the age-associated decline of antiviral immunity, 3-, 10-, and 22-month-old mice were treated with AED-sulfate (AED-S) for 45 days beginning 10 days prior to vaccination. Subsequently, mice were primed and boosted with suboptimal doses of a commercially-available trivalent influenza vaccine. Treatment of 10-month-old animals with AED-S during vaccination increased the titer of circulating antiviral immunoglobulin G to levels comparable with those in 3-month-old mice. Furthermore, AED-S treatment protected 10-month-old animals from intranasal challenge with a lethal dose of influenza virus 21 days after secondary vaccination. Although AED-S treatment of 22-month-old mice did not enhance vaccine responses and failed to protect against lethal challenge, the data from the 10-month-old animals suggest that treatment with AED-S will prevent the early manifestations of immunosenescence.

Evaluation of virus neutralization antibody levels in diabetic mutant mice.

Experiments were designed to determine whether genetic predisposition to diabetes or overt diabetes in the identical genotype had an effect on the level of neutralization antibodies to coxsackievirus B4 (CB4). Quantitation of neutralization antibody (NT) levels against CB4 was performed using both the 50% endpoint procedure and the multivariate Wilcoxon rank sum test. The results of these experiments demonstrate that the use of the multivariate procedure for the analysis of neutralization antibody levels provides both quantitative and qualitative information not evident when only the classical 50% end point procedure is used. Moreover, when comparison on antibody levels between different groups is being considered, the power of the comparison is markedly greater using the multivariate Wilcoxon rank sum test results. The present report provide an illustration of the difference in the qualitative and quantitative information obtained by the end-point procedure and the more comprehensive multivariate procedure for the analysis of neutralization antibody levels in diabetic mutant mice infected with coxsackievirus B4.

Genetic predisposition to diabetes mellitus is associated with impaired humoral immunity to coxsackievirus B4.

Experiments were performed to determine whether genetic predisposition to diabetes mellitus (DM) or clinical DM or both exert an influence on the production of neutralization antibodies to coxsackievirus B4 (CB4). The homozygous diabetic mutant mouse db+/db+, on the inbred C57BL/KsJ genetic background, develops a diabetes-like disease when maintained on ad libitum diet but restriction of excess food intake prevents overt disease. The doubly heterozygote db+/+m or the homozygote +m/+m misty coat color mutant, on the C57BL/KsJ genetic background, do not develop DM and served as controls. Animals infected with one-half a previously determined LD50 of CB4 were bled prior to infection and at 3, 5, 7, 14, 21 days and at 1, 2, 3, 4 and 5 months after infection. Serum neutralization antibody (NA) levels were determined from the percent CB4 plaque reduction. Until 2 months following infection, NA levels were not significant in either of the homozygous diabetic mutant groups, db+/db+. In the diabetic mutant group db+/db+, without overt disease, neutralization of CB4 when observed, was low, short-lived, and apparently not specific. However, in the homozygous diabetic mutants with spontaneous diabetes, CB4 NA became evident at 2 months after infection. By 3 months post-infection, serum NA levels were sufficient to cause 90% virus plaque reduction. These observations demonstrate that hereditary DM as characterized by the mutation diabetes, db, in the C57BL/KsJ mouse, is associated with a marked impaired humoral immune response to a diabetogenic human CB4. Specifically, there is an inability to develop an adequate level of anti-CB4 antibodies. The type and degree of immunological impairment are apparently different prior to and after onset of diabetes mellitus.

Reproduction of Pratylenchus penetrans on Potato and Crops Grown in Rotation with Potato.

The relative suitability of potato and crops frequently grown in rotation with potato as hosts for Pratylenchus penetrans was evaluated. Suitability of rye, wheat, corn, oat, sorgho-sudangrass, and potato were compared in pot studies based on ratios of final population : initial population density and densities of nematodes in roots at harvest. Population densities increased more on potato, oat, and corn than on rye, wheat, and sorgho-sudangrass. There were no differences among the four rye cultivars or between the two oat cultivars in host suitability. Population increases were not related to root weight or consistently to nematode densities in roots. Although rye and wheat were equally suitable hosts in pot studies, P. penetrans increased more on wheat than on rye in a field study, indicating that reproduction was reduced or mortality was increased on rye under field conditions.

Influence of Pratylenchus penetrans on Plant Growth and Water Relations in Potato.

Plants of potato (Solanum tuberosum) cultivars Katahdin and Superior were inoculated with 0, 1,500, or 15,000 Pratylenchus penetrans. Transpiration, measured in the greenhouse with a porometer after 56 days of growth, was not significantly different among nematode inoculum levels or between cultivars. The rate of xylem exudation from decapitated root systems of Katahdin plants inoculated with 1,500 or 15,000 P. penetrans and Superior plants inoculated with 15,000 P. penetrans was lower than from noninoculated plants. Root weight of Katahdin and Superior was not affected by P. penetrans inoculum level. Transpiration of plants inoculated with 0, 500, 5,000 or 50,000 P. penetrans was recorded weekly from 14 to 56 days after planting. No consistent effects of nematode inoculum density on transpiration rate were observed. Root hydraulic conductivity was lower in Katahdin plants inoculated with 266 P. penetrans per plant and in Chippewa with 5,081 per plant than in noninoculated plants. Nematodes reduced leaf area of Superior, Chippewa, and Katahdin and root dry weight of Chippewa but had no effect on growth of Hudson, Onaway, or Russet Burbank plants. Assessing nematode effects on root hydraulic conductivity may provide a measure of the tolerance of potato cultivars to nematodes.