The effect of salinity on enterocytozoon hepatopenaei infection in Penaeus vannamei under experimental conditions.

BACKGROUND: Enterocytozoon hepatopenaei (EHP) is an enteric pathogen that affects Penaeus vannamei and Penaeus monodon shrimp in many SE Asian countries. In the western hemisphere, EHP was reported for the first time in 2016 in farmed P. vannamei in Venezuela. Anecdotal evidence suggests that EHP is more prevalent in grow-out ponds where the salinity is high (> 15 parts per thousand (ppt)) compared to grow-out ponds with low salinities (< 5 ppt). Considering that P. vannamei is an euryhaline species, we were interested in knowing if EHP can propagate in P. vannamei in low salinities. RESULTS: In this study, we described an experimental infection using fecal strings as a source inoculum. Specific Pathogen Free (SPF) P. vannamei were maintained at three different salinities (2 ppt, 15 ppt, and 30 ppt) while continuously challenged using feces from known EHP-infected P. vannamei over a period of 3 weeks. The fecal strings, used as a source of EHP inocula in the challenges, was sufficient to elicit an infection in shrimp maintained at the three salinities. The infectivity of EHP in shrimp reared at 2 ppt, 15 ppt, and 30 ppt salinities was confirmed by PCR and histopathology. The prevalence and the severity of the EHP infection was higher at 30 ppt than at 2 ppt and 15 ppt. CONCLUSION: The data suggests that fecal strings are a reliable source of EHP inoculum to conduct experimental challenges via the fecal-oral route. An EHP infection can occur at a salinity as low as 2 ppt, however, the prevalence and the severity of the EHP infection is higher at a salinity of 30 ppt.

Retinoic acid syndrome--cardiac complication.

Retinoic acid syndrome is a novel complication of therapy with all-trans retinoic acid (ATRA) in patients with acute promyelocytic leukemia (APML). Primarily the syndrome consists of fever and respiratory distress. Additional features include weight gain, oedema over lower extremities, pleural or pericardial effusion and hypotension. We report electrophysiological changes in a 16 year old patient with acute promyelocytic leukemia following treatment with ATRA. Such an unusual complication is a rarity and to the best of our knowledge has not been previously reported.

A comparison of probabilistic and stochastic formulations in modelling growth uncertainty and variability.

We compare two approaches for inclusion of uncertainty/variability in modelling growth in size-structured population models. One entails imposing a probabilistic structure on growth rates in the population while the other involves formulating growth as a stochastic Markov diffusion process. We present a theoretical analysis that allows one to include comparable levels of uncertainty in the two distinct formulations in making comparisons of the two approaches.

Rapid plant regeneration and analysis of genetic fidelity of in vitro derived plants of Chlorophytum arundinaceum Baker--an endangered medicinal herb.

An efficient in vitro multiplication system via multiple shoot bud induction and regeneration has been developed in Chlorophytum arundinaceum using shoot crown explants. Optimum regeneration frequency (87%) and desirable organogenetic response in the form of de novo organized multiple shoot buds without an intervening callus phase was obtained on Murashige and Skoog's (MS) minimal organics medium containing 3% sucrose (w/v) supplemented with 4 x 10(-6) M Kn and 2 x 10(-6) MIBA. Axenic secondary explants with multiple shoot buds on subculturing elicited best response with 1 x 10(-5) M Kinetin (Kn) and 5 x 10(-6) M indole-3-butyric acid (IBA) giving rise to an average of 18.74 shoots per culture with mean shoot length of 7.6 cm +/- 1.73. Varying molar ratios of either Kn/IBA or Kn/NAA revealed statistically significant differences in the regeneration frequencies among the phytohormone treatments. It was observed that the shoot bud differentiation and regeneration was influenced by the molar ratios of cytokinins/auxin rather than their relative concentrations. Healthy regenerated shoots were rooted in half strength MS basal medium containing 3% sucrose (w/v) supplemented with 5 x 10(-6) M IBA. Following simple hardening procedures, rooted plantlets, were transferred to soil-sand (1:1; v/v) with more than 90% success. Genetic fidelity was assessed using random amplified polymorphic DNA (RAPD), karyotype analysis and meiotic behaviour of in vitro and in vivo plants. Five arbitrary decamers displayed same banding profile within all the micropropagated plants and in vivo explant donor. The cytological and molecular analysis complemented and compared well and showed no genomic alterations in the plants regenerated through shoot bud differentiation. High multiplication frequency, molecular, cytological and phenotypic stability ensures the efficacy of the protocol developed for the production and conservation of this important endangered medicinal herb.

Cytomixis impairs meiosis and influences reproductive success in Chlorophytum comosum (Thunb) Jacq. - an additional strategy and possible implications.

Spontaneous intercellular chromatin migration/cytomixis was observed to occur in the pollen mother cells (PMCs) of the Chlorophytum comosum for the first time. The migration through cytomictic channels was more pronounced in meiosis-I and very rare in meiosis-II. The process was associated with erratic meiosis, which was characterized by defects in chromosome organization and segregation. Cytomixis was more intense in the month of April than in July and consequently the frequency of meiotic irregularities was much more pronounced during the month of April. As a consequence of abnormal meiosis, fertility was drastically reduced resulting in meager seed efficiency of 17% only. Recombination system also does not guarantee the release of sufficient variability. We view the phenomenon of cytomixis as genetically controlled mechanism involving meiotic genes and operating through signal transduction pathway triggered by the environmental stimuli. The evolutionary significance and tenable hypothesis in the backdrop of existing literature is also proposed.

Modeling shrimp biomass and viral infection for production of biological countermeasures.

In this paper we develop a mathematical model for the rapid production of large quantities of therapeutic and preventive countermeasures. We couple equations for biomass production with those for vaccine production in shrimp that have been infected with a recombinant viral vector expressing a foreign antigen. The model system entails both size and class-age structure.

Identification of differentially expressed genes in shrimp (Penaeus stylirostris) infected with White spot syndrome virus by cDNA microarrays.

White spot syndrome virus (WSSV) is currently the most important viral pathogen infecting penaeid shrimp worldwide. Although considerable progress has been made in characterizing the WSSV genome and developing detection methods, information pertaining to host genes involved in WSSV pathogenesis is limited. We examined the potential of cDNA microarray analysis to study gene expression in WSSV-infected shrimp. Shrimp cDNAs were printed as low-density arrays on glass slides and were hybridized with Cy3/Cy5 labeled probes derived from RNA isolated from healthy and WSSV-infected shrimp. Genes that code for proteins that are relevant to crustacean immunity, structural proteins, as well as proteins of unknown function were among those whose mRNA expression was altered upon WSSV infection. To validate the microarray data, the temporal expression of three differentially expressed genes, an immune gene (C-type lectin-1), a structural gene (40S ribosomal protein), and a gene involved in lipid metabolism (fatty acid binding protein) was measured in healthy and WSSV-infected shrimp by real-time RT-PCR. The data suggest that WSSV infection alters the expression of a wide array of cellular genes, and provides a framework for further studies aimed at identifying genes whose function may provide insight into the mechanism of WSSV infection in shrimp.

Isolation of differentially expressed genes from white spot virus (WSV) infected Pacific blue shrimp (Penaeus stylirostris).

To isolate novel cellular factors that are activated or repressed upon WSV infection, the RNA fingerprints of healthy and WSV infected blue shrimp ( Penaeus stylirostris) were compared using the mRNA differential display technique. Thirty-two unique differentially expressed, and one constitutively expressed, cDNA sequences were retrieved. Six of 32 cDNAs showed similarities with the database entries: cDNA 10G32-142 to a shrimp arginine kinase, 22C48-201 to shrimp mitochondrial ATPase gene; 22C47-197, 21G49-203 and 20A55-268 to shrimp ESTs and 20G50-206 to a WSV gene, ORF 116. The constitutively expressed gene showed significant similarity to a yeast elongation factor 1-alpha gene. The expression of a subset of differentially expressed genes (13 of 32) was further evaluated by real-time RT-PCR. Ten of 13 genes showed statistically significant changes in expression between healthy and WSV infected animals suggesting that these genes may play an important role in WSV pathogenesis.

Detection and quantification of infectious hypodermal and hematopoietic necrosis virus and white spot virus in shrimp using real-time quantitative PCR and SYBR Green chemistry.

A rapid and highly sensitive real-time PCR detection and quantification method for infectious hypodermal and hematopoietic necrosis virus (IHHNV), a single-stranded DNA virus, and white spot virus (WSV), a double-stranded DNA (dsDNA) virus infecting penaeid shrimp (Penaeus sp.), was developed using the GeneAmp 5700 sequence detection system coupled with SYBR Green chemistry. The PCR mixture contains a fluorescence dye, SYBR Green, which upon binding to dsDNA exhibits fluorescence enhancement. The enhancement of fluorescence was proportional to the initial concentration of the template DNA. A linear relationship was observed between the amount of input plasmid DNA and cycle threshold (C(T)) values over a range of 1 to 10(5) copies of the viral genome. To control the variation in sampling and processing among samples, the shrimp beta-actin gene was amplified in parallel with the viral DNA. The C(T) values of IHHNV- and WSV-infected samples were used to determine absolute viral copy numbers from the standard C(T) curves of these viruses. For each virus and its beta-actin control, the specificity of amplification was monitored by using the dissociation curve of the amplified product. Using genomic DNA as a template, SYBR Green PCR was found to be 100- to 2000-fold more sensitive than conventional PCR, depending on the virus, for the samples tested. The results demonstrate that SYBR Green PCR can be used as a rapid and highly sensitive detection and quantification method for shrimp viruses and that it is amenable to high-throughout assay.

Nucleotide sequence of 3'-end of the genome of Taura syndrome virus of shrimp suggests that it is related to insect picornaviruses.

Taura syndrome disease, caused by Taura syndrome virus (TSV), is one of the most important viral diseases of penaeid shrimp in the Western Hemisphere resulting in catastrophic disease epidemics in farmed shrimp. We have cloned and sequenced a 3278 bp cDNA representing the 3' end of the TSV genome. Sequence analyses revealed that frame + 2 had the longest open reading (ORF) frame. This frame contained a 5'-terminal 19 non-coding bases followed by an ORF from nucleotides 20 to 3053 (encoding 1011 amino acids, aa) and a 3' untranslated region of 225 nts. The deduced aa sequence of TSV showed significant similarities with those of the coat proteins of insect picornaviruses, Rhopalosiphum padi virus, Plautia stali intestine virus, Drosophila C virus, Triatoma virus of Triatoma infestans and Himetobi P virus of brown plant hopper. A single transcript of approximately 10 kb was detected by Northern blot hybridization suggesting that the TSV coat protein gene is not expressed as a subgenomic RNA. We concluded that the genome organization of TSV is similar to insect picornaviruses. This is the first molecular evidence of occurrence of a picornavirus in the class Decapoda.

Age and nortriptyline concentrations in plasma ultrafiltrate.

OBJECTIVES: Since plasma protein binding of tricyclic antidepressants may be relevant to treatment effects and can be influenced by age-associated factors, we examined both plasma ultrafiltrate and total concentrations of nortriptyline (NT) in patients and compared these to age. We hypothesized negative associations with age of both ultrafiltrate NT and the ratio of ultrafiltrate NT to total plasma NT. METHODS: Patients with major depression at a psychiatric service treated with a stable dose of NT were studied. Trough plasma ultrafiltrate NT concentrations and total plasma NT concentrations were measured by high performance liquid chromatography. Concentrations were corrected for dose. RESULTS: Eighty-seven patients aged 26 - 88 years were studied. Ultrafiltrate NT concentrations and the ratio of ultrafiltrate NT to total NT concentrations were both significantly negatively associated with age. Total NT concentrations were not significantly associated with age. CONCLUSIONS: Relatively low ultrafiltrate NT concentrations in older patients may reflect lower tissue exposure at a given total plasma NT concentration. This could be relevant to toxic and therapeutic effects. Studies of relationships between non-bound drug concentrations and NT treatment outcomes across the age span are needed.

Infectious hypodermal and hematopoietic necrosis virus of shrimp is related to mosquito brevidensoviruses.

We purified and sequenced infectious hypodermal and hematopoietic necrosis virus (IHHNV), a small DNA virus of shrimp, from wild Penaeus stylirostris. The virion has a buoyant density of 1.45 as determined by cesium chloride gradient. Analysis of 3873 nucleotides of the viral genome revealed three large open reading frames (ORFs) and parts of the noncoding termini of the viral genome. The left, mid, and right ORFs on the complementary (plus) strand have potential coding capacities of 666 amino acids (aa) (75.77 kDa), 363 aa (42.11 kDa), and 329 aa (37.48 kDa), respectively. The overall genomic organization is similar to that of the mosquito brevidensoviruses. The left ORF most likely encodes the major nonstructural (NS) protein (NS-1) since it contains conserved replication initiator motifs and NTP-binding and helicase domains similar to those in NS-1 from all other parvoviruses. The IHHNV putative NS-1 shares the highest aa sequence homology with the NS-1 of mosquito brevidensoviruses, Aedes densovirus and Aedes albopictus parvovirus. A search for putative splicing sites revealed that the N-terminal region of NS-1 is very likely located in a small ORF upstream of the left ORF. The right ORF is presumed to encode structural polypeptides (VPs), as in other parvoviruses. Two putative promoters, located upstream of the left and right ORFs, are presumed to regulate expression of NS and VP genes, respectively. Thus, IHHNV is closely related to densoviruses of the genus Brevidensovirus in the family Parvoviridae, and we therefore propose to rename this virus Penaeus stylirostris densovirus (PstDNV).

Determination of the cDNA sequence and mRNA expression of interleukin-1 receptor antagonist in horses.

OBJECTIVE: To determine the complementary DNA (cDNA) sequence of interleukin-1 receptor antagonist (IL-1ra) in horses and compare messenger RNA (mRNA) expression of IL-1ra among horses of various breeds. SAMPLE POPULATION: Blood samples from neonatal and adult horses examined for a variety of diseases. PROCEDURE: A polymerase chain reaction procedure was used to amplify a 220 base pair (bp) portion of the genomic DNA. The upstream and downstream regions of the cDNA sequence were determined by means of 5' and 3' rapid amplification of cDNA ends (RACE) procedures. Northern blot hybridization was used to examine steady-state mRNA expression of IL-1ra. RESULTS: The consensus sequence of the cDNA obtained with the 5'-RACE procedure and the sequence for the 220 bp portion of the genomic DNA represented the putative sequence for secreted IL-1ra. The predicted secreted IL-1ra amino acid sequence contained 176 residues with an in-frame stop codon; the N-terminal 25 amino acid residues resembled the signal peptide reported for human secreted IL-1ra. An approximately 1.3 kilobase pair (kb) band that represented a portion of the 3' end of the coding region and the 3' untranslated region was obtained by use of the 3' -RACE procedure. Northern blot hybridization detected a 1.6 kb transcript in blood RNA from adult Arabian, Belgian, Thoroughbred, and Standardbred horses. CONCLUSIONS: Results suggest that the DNA for equine secreted IL-1ra has a short (29 bp) 5' untranslated region, a 534 bp coding region, and a long (approximately 1,080 bp) untranslated region.

Identification of abundant and informative microsatellites from shrimp (Penaeus monodon) genome.

Microsatellites were isolated from P. monodon genomic libraries by direct sequencing of recombinant clones without probe screening. Forty-nine out of 83 clones sequenced contained 99 microsatellite arrays of three or more repeats. When five or more and ten or more repeats were considered, 28 and 14 microsatellites were detected, respectively. The 99 microsatellites were classified as perfect (75%), imperfect (6%), compound perfect (3%) and compound imperfect (16%). The abundance of di-, tri-, tetra- and hexanucleotide repeats were 67%, 20%, 9% and 3%, respectively. The dinucleotide repeats included 36 (CT)n, 31 (GT)n, 17(AT)n and 3 (CG)n. One octanucleotide repeat (ATTTATTC)5 was found within a large repeat sequence. Optimal annealing temperatures were determined for PCR using 11 primer sets encompassing 15 microsatellites. Ten primer sets provided successful amplifications with allele sizes generally ranging from 139 to 410 bp. All these primers amplified polymorphic loci with PIC values ranging from 0.63 to 0.96. Two primer sets amplified additional bands which can easily be distinguished from the bands of the main locus. Three out of 10 P. monodon microsatellites also amplified alleles in P. vannamei. The abundance and informative nature of P. monodon microsatellites and their potential for cross-species amplification make them useful for genetic studies.

Molecular characterization of coat protein genes of serologically distinct isolates of potato virus Y necrotic strain.

The cost protein (CP) genes of two potato virus Y necrotic isolates (N27 and a mutant strain N27-92), which differed in their reactivity to a monoclonal antibody (mab), were characterized. Both isolates could be detected by mab 4E7, but mab VN295.5 selectively reacted to N27 and not to N27-92. The CP genes of both isolates coded for 267 amino acids with approximately 99.0% identity at both the nucleotide and the amino acid levels. nucleotide sequence comparison indicated five substitutions in N27-92 compared with N27. Three of these changes resulted in substitution of amino acids. Two transitions (A-->G) in N27-92 changed threonine to alanine and lysine to arginine at positions 7 and 55, respectively, whereas a A-->T transversion changed asparagine to isoleucine at positions 27. The surface probability curves of both the isolates could almost be superimposed, except at amino acid positions 7 and 27. Since amino acid substitution at position 55 is conservative, changes from polar to hydrophobic amino acids (threonine-->alanine and asparagine-->isoleucine) at positions 7 and 27 might have changed the epitope(s) of N27-92, abolishing its detection by mab VN295.5.