French vertical-flow constructed wetland design: adaptations for tropical climates.

The French Outermost Regions are under tropical climate yet still have to comply with both French and EU regulations. French vertical-flow constructed wetland systems appear well adapted to the technical specifics of these regions but their adaptation to tropical climate requires new design guidelines to be defined (area needed, number of filters, type of plants, material to be used, etc.). A study was started in 2008, with backing from the national water authorities, to implement full-scale experimental sites and assess the impacts of local context on design and performances. This paper reports the monitoring results on three vertical-flow constructed wetlands fed directly with raw wastewater (known as the 'French system') in Mayotte and French Guiana. The plants, now in operation for between 1 and 6 years, range from 160 to 480 population equivalent (p.e.). Monitoring consisted of 28 daily composite flow samples in different seasons (dry season, rainy season) at the inlet and outlet of each filter. Performances are benchmarked against French mainland area standards from Irstea's database. Results show that performances are improved by warmer temperature for chemical oxygen demand (COD), suspended solids (SS) and total Kjeldahl nitrogen (TKN) and satisfy national quality objectives with a single stage of filters. Treatment plant footprint can thus be reduced as only two parallel filters are needed. Indeed, warm temperatures allow faster mineralization of the sludge deposit, making it possible to operate at similar rest and feeding period durations. Systems operated using one twin-filter stage can achieve over 90% COD, SS and TKN removal for a total surface of 0.8 m²/p.e.

Efficacy of 1,3-dichloropropene in soil amended with compost and unamended soil.

1,3-Dichloropropene (1,3-D) is a likely alternative soil fumigant for methyl bromide. The objective was to determine root-knot nematode, Meloidogyne incognita, survival in microplots after exposure to 1,3-D for various periods of time in soil that have previously been amended with compost. The treatments were 1,3-D applied broadcast at 112 liters/ha and untreated controls in both compost-amended and unamended soil. Soil samples were collected from each microplot at 6, 24, 48, 72, and 96 hours after fumigation at three depths (0-15, 15-30, and 30-45 cm). One week after fumigation, six tomato seedlings were transplanted into each microplot and root galling was recorded 6 weeks later. Plants grown in fumigated compost-amended soil had more galls than plants from fumigated unamended soil at P

Chicken Litter Soil Amendment Effects on Soilborne Microbes and Meloidogyne incognita on Cotton.

The effects of chicken litter incorporated 28, 14, and 0 days before planting on Meloidogyne incognita in cotton and soil organisms were determined in the greenhouse. Treatments consisted of field soil amended with litter at rates of 0, 0.125, 0.25, 0.5, and 1% by weight. At 45 and 90 days after planting, numbers of M. incognita decreased as rates of litter increased. Microbivorous nematode densities increased as litter rates increased only in the first experiment. Plant growth increased as litter rates increased, regardless of when the litter was incorporated, or the presence or absence of M. incognita. Bacterial and fungal CFU fluctuated during both experiments, but generally had positive linear relationships with litter rate. Population densities of M. incognita decreased with increasing bacterial and fungal counts in amended soil. Bacterial genera identified from the litter-amended soil included Arthrobacter, Bacillus, Cellulomonas, Mi-crococcus, Pseudomonas, and Rhodococcus.

Rate response of 1,3-dichloropropene for nematode control in spring squash in deep sand soils.

The soil fumigant 1,3-dichloropropene (1,3-D) formulated with chloropicrin is viewed as a likely alternative for replacing methyl bromide in Florida when the latter is phased out in 2005. Therefore, it behooves us to learn more about using 1,3-D in deep, sand soils. Two trials were conducted on spring squash to determine the most effective rate of 1,3-D for the control of Meloidogyne spp. Rates tested included 0, 56, 84, 112, and 168 liters/ha of 1,3-D applied broadcast with conventional chisels 30 cm deep. The chisel traces were sealed by disking immediately after fumigant application. Cucurbita pepo cv. Sunex 9602 was sown 7 days after fumigation. The population density of plant-parasitic nematodes in soil and root-knot nematode galling severity was determined at 34 and 65 days after planting (DAP), and the number of marketable fruit and yield were determined. The number of fruit and yield were higher in all plots that received 1,3-D than in untreated controls. The number of Meloidogyne spp. second-stage juveniles was lower in all fumigated plots in trial 1 at both 34 and 65 DAP, and in trial 2 at 65 DAP, than in the untreated control. The severity of root galling was decreased with all treatments in both trials, with broadcast rates of 84, 112, and 168 liters/ha providing the best control of root-knot nematodes in spring squash grown in sandy soil. Satisfactory management of root knot on squash grown in early spring months in north Florida can be achieved with low rates of 1,3-D.

Characterization of the replication cycle of the Lymantria dispar nuclear polyhedrosis virus.

The life cycle of the Lymantria dispar nuclear polyhedrosis virus (LdMNPV) was characterized through analysis of budded virus (BV) release, the temporal formation of polyhedra, the temporal transcription pattern of representative early, late, and hyper-expressed late genes, and the onset of DNA replication in the Ld652Y cell line. Transcripts from the LdMNPV immediate early gene G22 were detected 4 h post infection (h p.i.). The late and hyper-expressed late p39 capsid and polyhedrin genes were initially transcribed at approximately 20 and 24 h p.i., respectively. Viral DNA replication initiated at approximately 18-20 h p.i. Budded virus was released from infected cells between 24 and 36 h p.i., and polyhedra were first detected at approximately 48 h p.i.

Meloidogyne incognita Infested Soil Amended With Chicken Litter.

The effects of chicken litter on Meloidogyne incognita in cotton, Gossypium hirsutum cv. DPL50 were determined in field microplots. Litters (manure and pine-shaving bedding) from a research facility and a commercial broiler house were used. Treatments consisted of 0.25%, 0.5%, and 1% litter by dry weight of soil for each kind of litter. Three control treatments consisted of soil not amended with litter, with and without nematodes, and one treatment to which mineral fertilizer was added at a nitrogen rate equivalent to that of the 0.5% litter rate, with nematodes. Microplots were inoculated at planting with 900 eggs/100 cm(3) soil in 1993 and 1,000 eggs/100 cm(3) soil in 1994. At 92 and 184 days after planting, nematode population densities decreased linearly with increasing rates of litter. Nematode numbers at midseason were larger in plots treated with mineral fertilizer than in plots treated with a rate of litter equivalent to the 0.5% rate. Fungal and bacterial population densities fluctuated throughout the growing season. Bacterial numbers had a positive linear relationship, with increasing rates of litter only in October 1993; however, significant positive relationships were observed throughout the 1994 growing season. In 1994, nematode population density at 92 days after planting decreased linearly with increasing bacterial numbers 30 days after planting. No other significant relationships between nematode densities and microbial densities were observed. Fungi and bacteria isolated from the litter and litter-amended soil were identified. Fungal genera isolated included Acremonium, Aspergillus, Eurotium, Paecilomyces, Petriella, and Scopulariopsis, whereas bacteria genera included Arthrobacter, Bacillus, and Pseudomonus.

Identification and characterization of the ecdysteroid UDP-glucosyltransferase gene of the Lymantria dispar multinucleocapsid nuclear polyhedrosis virus.

We have located, cloned, sequenced and characterized the ecdysteroid UDP-glycosyltransferase gene (egt) gene from the baculovirus Lymantria dispar multinucleocapsid nuclear polyhedrosis virus. (LdMNPV), which is specific for the gypsy moth (L. dispar). The egt gene from the related baculovirus Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) disrupts the hormonal balance of the host larva by galactosylating ecdysone, which prevents moulting. The location of the LdMNPV egt gene, determined by hybridization analysis using a cloned coding segment of the AcMNPV egt gene, was mapped to between 79.1 and 80.2 map units on the viral genome. This region contains an open reading frame of 1464 nucleotides capable of encoding a 55K polypeptide. This predicted protein exhibits a 42% amino acid identity with the AcMNPV egt polypeptide. Transcripts of the egt gene were analyzed by Northern blot and primer extension. The egt gene is transcribed from approximately 12 to 48 h, and maximally at about 16 h post-infection. Transcription occurred in the presence of aphidicolin, a viral DNA synthesis inhibitor, but not in the presence of cycloheximide, a protein synthesis inhibitor. Therefore the LdMNPV egt gene is classified as a delayed early gene. The egt gene is transcribed in a clockwise direction with respect to the circular map, and transcription initiates at a single site. Comparisons between the two baculoviral egt proteins and mammalian UDP-glucuronosyltransferases reveal areas which are conserved between the mammalian and baculoviral genes, as well as areas that are only conserved in the viral egt proteins. The LdMNPV protein sequence appears to include a signal peptide, which would allow the protein to be secreted into the haemolymph.

Epitope specificity of the bovine antibody response to the gIII glycoprotein of bovine herpesvirus type 1.

A panel of monoclonal antibodies (MAbs) to BHV-1, specific for gI and gIII glycoproteins and for a nonglycosylated core protein p100, was used to examine the epitope specificity of the immune response to the virus in naturally exposed and experimentally infected cattle. Sera from experimentally infected calves were analyzed in a competition ELISA (C-ELISA). Antibody to an epitope on gIII appeared as early as 4 days post infection, although virus-neutralizing antibody did not appear until day 8 or later. Antibody production peaked at 13 to 18 days post infection but the level of antibody to each gIII epitope varied. Competition by sera from cattle naturally exposed to BHV-1 was analyzed as a function of the virus neutralization (VN) titer of these sera. Competition with most of the MAbs correlated linearly with neutralization titer. However, competition with 2 of the MAbs, one specific for gIII and one specific for gI, was maximal even at the lowest neutralization titer, and competition with another MAb, specific for a non-glycosylated core protein, p100, was negative. Selected sera from the naturally exposed group were also examined by radioimmunprecipitation, and were shown to react predominantly with gI, gIII and gIV glycoproteins and in a few shown to react predominantly with gI, gIII and gIV glycoproteins and in a few MAbs were determined, and it was found that neutralization was enhanced by certain combinations. A mutually exclusive relationship between neutralization enhancement and competition for binding by MAbs (as determined by reciprocal C-ELISA) indicated an epitope-specific, rather than antibody-specific, mechanism for neutralization enhancement.

Rapid, sensitive, competitive serologic enzyme-linked immunosorbent assay for detecting serum antibodies to bovine herpesvirus type 1.

An enzyme-linked immunosorbent assay (ELISA) for the detection of cattle antibodies to bovine herpesvirus type 1 was developed on the basis of competition between serum antibody and a virus-neutralizing mouse monoclonal antibody. The assay showed improved sensitivity over the virus neutralization (VN) test and over an enhanced VN test in which incubation of antibody-virus mixtures was carried out for 24 h. With the ELISA, antibodies in sera from experimentally infected cattle were detected earlier after infection and showed more rapid increases in levels. A comparison of the ELISA with the VN tests by using a set of 85 field sera with low levels of antibodies demonstrated that the ELISA was the most sensitive test, detecting 10 positive serum samples that were negative by the VN tests. The ELISA was inexpensive, rapid, and highly reproducible and showed a significant improvement in sensitivity over VN tests.

Shedding of enteric coronavirus in adult cattle.

Electron microscopic examination of fecal specimens from adult dairy cows indicated seasonal coronavirus shedding. Fifty-two cows from a 300 cow herd were monitored for shedding of coronavirus. Approximately 50% to 60% of the cows monitored shed coronavirus during the winter months (November to March) of the first year of the study. During 2 subsequent years of monitoring the same cows, 20% to 30% of the cows shed coronavirus during the winter months. Virus shedding was not detected during the summer months (July to September). Half of the cows monitored were vaccinated with a modified-live rotavirus-coronavirus-Escherichia coli combination vaccine; however, vaccination did not influence seasonal shedding of coronavirus, as compared with shedding in the nonvaccinated cows. In nonvaccinated cows that calved in the winter months, the incidence of coronavirus shedding increased from 20% to 30% during the last 2 months of gestation to 65% to 70% at parturition. In vaccinated cows, the incidence of shedding did not increase at parturition.

Immune response to bovine herpes herpesvirus type 1 infections: virus-specific antibodies in sera from infected animals.

The virus specificity of antibodies against bovine herpes virus type 1 was determined with a radioimmunoprecipitation assay and serum collected from natural and experimentally induced infections. By using sequentially collected sera, the development of antibodies to 4 to 5 viral glycoproteins and 11 to 12 nonglycosylated proteins was followed for the first 50 days after infection. The major and most consistent responses in experimentally and naturally infected animals were to four glycoproteins with molecular weights of 102,000, 96,000, 69,000, and 55,000, as well as to a major virion 115,000-molecular-weight nonglycosylated protein. The four glycoproteins were all coprecipitated by a neutralizing monoclonal antibody and were probably involved as target antigens in virus neutralization. Another antigenically unrelated glycoprotein with a molecular weight of 82,000 and a nonglycosylated protein with a molecular weight of 91,000 were also precipitated, but the immune response to these two proteins was transient. Reactivity to gp82 was only weakly detected in serum from naturally infected animals. Contact control animals which did not contract a bovine herpes virus type 1 infection but were exposed to infected animals with signs of severe illness had antibodies which recognized gp102, gp96, gp69 and gp55 as well as p115. These antibodies were present in low amounts and, in contrast to infected animals, did not increase between acute and convalescent sampling.

A single dilution enzyme-linked immunosorbent assay for the quantitative detection of antibodies to bovine herpesvirus type 1.

Three serological assays were compared for detection of antibodies to bovine herpes-virus type 1. These were virus neutralization (VN), enhanced complement fixation (CF) and enzyme-linked immunosorbent assay (ELISA). The ELISA was developed using an infected cell lysate antigen and purified virus and was optimized in relation to antigen and antisera dilutions. The CF assay was enhanced by the addition of bovine complement. These 3 assays were compared for detection of: specific virus antibody titers; sero-conversions; early antibody response in experimentally-infected cattle. Both ELISA end-point titers and single dilution values were found to be more sensitive than the CF or VN assays for specific antibody level quantitation. With a single dilution ELISA test procedure a correlation was obtained between ELISA values and VN titers. Using the single dilution ELISA test the assay also detected antibodies in experimentally-infected cattle before either the VN or CF assays, and agreed with the VN test in 35/38 seroconversions found by 4-fold or more VN changes between acute and convalescent paired sera from naturally-infected animals. The single dilution ELISA was a rapid and sensitive test for routine antibody detection in bovine sera.

Neutralizing determinants defined by monoclonal antibodies on polypeptides specified by bovine herpesvirus 1.

Monoclonal antibodies were used to study neutralizing determinants on polypeptides of bovine herpesvirus 1. Two of three monoclonal antibodies which recognized nonoverlapping epitopes on a glycoprotein of 82,000 daltons were found to neutralize. A second group of monoclonal antibodies that individually precipitated five viral glycopolypeptides ranging in size from 102,000 to 55,000 daltons also neutralized. Two monoclonal antibodies which were the most efficient in neutralization recognized a non-glycosylated protein of 115,000 daltons which was the major polypeptide on the virus. A fourth group of monoclonal antibodies precipitated a non-glycosylated polypeptide of 91,000 daltons and several smaller polypeptides, but these antibodies demonstrated only limited neutralizing activity.

A micro cell culture method utilizing modified microscope slides for use in fluorescent antibody and enzyme immunoassays.

A technique for culturing small quantities of mammalian cells on modified microscope slides is described. The modified microscope slides were Bellco Glass, Inc., toxoplasmosis slides and the cell cultures used were early passage bovine embryonic lung cells and continuous cell lines of porcine and canine origins. The slide cell cultures were either uninfected or infected with selected viruses or the obligate intracellular protozoanEncephalitozoon caniculi for utilization in direct and indirect fluorescent antibody testing or in peroxidase antiperoxidase immunosorbant assays.