Quantitative extraction of macro-invertebrates from temperate and tropical leaf litter and soil: efficiency and time-dependent taxonomic biases of the Winkler extraction

Winkler extractors, a simple device presumed to extract macro-invertebrates efficiently from soil and litter samples, is being used increasingly in ecological surveys and functional studies of soil macro-invertebrate communities. In this study the extraction efficiency and taxonomic bias of the Winkler extraction are evaluated for extraction periods of 3 h up to 7 weeks, calibrated by hand-sorting after 7 weeks. The method extracts most macro-invertebrates completely or to a proportion of over 90% except Isopoda, Diplopoda and Mollusca. However, for an exhaustive result, a long extraction period of several weeks is necessary. For the most speciose group (adult beetles) and for the commonly most abundant group (ants), a short extraction of 3 days was sufficient to get 70% of the individuals and nearly all species. Three days was also sufficient to recover the rank abundance order of beetle families, while for ‘higher taxa’ and for Chilopoda species, 4 and 3 weeks were necessary, respectively. Optimum extraction times for the abundant macro-invertebrate groups and possible adjustment factors for the soil macro-invertebrates of temperate woodlands are proposed to compensate the taxonomic bias caused by short extraction periods. However, for recording an accurate snapshot of the soil and litter fauna at a particular time, shorter extraction periods are advisable because of the short life cycle of many soil invertebrates causing emergence of later stages or a second generation during longer extraction periods. The problem of contamination of samples is also discussed.     

Vaccination of chickens with a recombinant fowlpox virus containing the hemagglutinin-neuraminidase gene of Newcastle disease virus under the control of the fowlpox virus thymidine kinase promoter.

When chickens were vaccinated with a recombinant fowlpox virus (FPV) containing the Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) cDNA under the control of the thymidine kinase (TK) promoter and inserted into the FPV TK gene, the FPV antibody response to the recombinant virus was similar to the response to vaccination with standard FPV, and the recombinant virus protected chickens against challenge with virulent FPV. While the presence of the NDV HN cDNA was demonstrated in the recombinant virus, which was stable on serial passage, expression of HN was not detected by hemagglutination, Western blot analysis or immunoprecipitation of infected cell lysate. Chickens vaccinated with the recombinant virus failed to mount an NDV hemagglutination-inhibition antibody response, and they did not resist challenge with velogenic NDV. It was concluded that the TK promoter was too weak to drive the HN gene, but that the insertion into the FPV TK gene did not reduce the immunogenicity of the virus.     

Characterization of fowl adenoviruses isolated in Ontario and Quebec, Canada

Fowl adenoviruses (FAdV) are generally considered ubiquitous, but certain serotypes and strains are known to be associated with primary diseases, such as inclusion body hepatitis (IBH). Fifty-two FAdV isolates were collected from the provinces of Ontario and Quebec over a 4-year period. These 2 provinces have the largest poultry industries in Canada. Except for one virus, which originated from a guinea fowl, all other viruses were isolated from chicken samples. Most of these were from broilers, although some were from broiler breeders, and one was from layer pullets. Thirty-four isolates were from clinical IBH cases with the final laboratory diagnosis of IBH; however, for 18 isolates, the varied case diagnosis was seemingly unrelated to FAdV. All IBH-associated viruses had deoxyribonucleic acid (DNA) profiles compatible with FAdV species E (28 cases) or species D (6 cases), and the DNA fragment profiles of 26 species E viruses were indicative of serotype 8. Two viruses were serotype 6, as confirmed by virus neutralization. All species D viruses had a DNA profile similar to that of FAdV-2. The number of serotype 8 virus isolations has increased over the years, and by 2001 serotype 8 had become the dominant serotype in Ontario, and continues to be so. Moreover, this virus (FAdV-8) has shown a strong association with IBH.     

Comparison of Estimated Costs and Benefits of Site-Specific Versus Uniform Management for the Bean Leaf Beetle in Soybean

Six soybean, Glycine max (L.) Merrill, fields were examined to compare estimated costs and benefits for uniform and site-specific management (SSM) programs for the bean leaf beetle, Cerotoma trifurcata (Forster). Beetle counts and soybean yield were field-collected, insecticide and sampling costs were estimated. In five fields, site-specific management produced only slightly greater return. The inclusion of sampling costs in each scenario resulted in higher return for the uniform scenario. The uniform management scenario estimated pest pressure as well as the site-specific scenario when beetle populations were high or low. In one field with moderate pest pressure, the SSM scenario would have increased insecticide use. The estimations in this study are based on hypothetical scenarios and the application equipment to target insecticides based on map coordinates is not readily available. The economic estimations provide examples of current limitations of site-specific management that need to be addressed before this technology becomes valuable for soybean insect management.     

Antibody detection-based differential ELISA for NDV-infected or vaccinated chickens versus NDV HN-subunit vaccinated chickens

With the advent of subunit vaccines for microbial diseases it is becoming increasingly important to be able to differentiate naturally infected animals from those vaccinated with the corresponding subunit vaccine. For avian viruses such as Newcastle disease virus (NDV), a whole virus-based ELISA cannot make such a differential diagnosis since in both cases the antisera would react with the whole virus. The nucleocapsid protein (NP) gene of the NDV Hitchner B1 strain was cloned, sequenced and expressed to develop a differential ELISA. The B1 NP had 95.7 and 96.1% amino acid identities with the NP of the d26 and Ulster 2C strains, respectively. The B1 NP expressed in a baculovirus expression vector (recNP) was the expected size and reacted with NDV-specific antibodies (Ab) in Western blots and by radioimmunoprecipitation. The ELISA using recNP-coated wells, tested on serum samples from flocks pretested with a commercial NDV kit gave results corresponding to those of the kit. Furthermore, use of both the renNP-based ELISA and a whole virus ELISA allowed the differentiation of birds vaccinated and a NDV haemagglutinin-neuraminidase (HN) expressing fowlpox virus from birds infected with NDV. This provides the basis for establishing an ELISA that discriminates between the antibody response to a recombinant fowlpox vaccine (expressing NDV HN protein) and that to live and inactivated NDV.     

Mapping of porcine parvovirus DNA and development of a diagnostic DNA probe

Dimeric and monomeric replicative forms of DNA of porcine parvovirus (PPV) strain NADL-2 were isolated and examined by restriction enzyme analysis and reciprocal Southern blot hybridization during development of a DNA probe for PPV. Genomic single stranded PPV DNA was 5.0 kb long, and results substantiated the rolling-hairpin model of parvovirus DNA replication with the primer sequence located in the 3' terminal hairpin loop. An additional finding was the generation of a 4.7 kb species of viral DNA which was considered to be a 0.3 kb deletion variant of genomic PPV DNA. A 3.0 kb DNA fragment obtained by Pst I/Hind III digestion of monomer replicative form DNA was cloned into a plasmid vector, pUC 19. The cloned fragment, recovered from transformed Escherichia coli strain TB1 and labelled with [32P] dCTP, was evaluated by dot hybridization as a probe for PPV in infected cell cultures. The probe was specific for PPV infected cells, and was 100 times more sensitive than the standard hemagglutination test.     

Indirect method for prediction of hemagglutination inhibition antibody titers to Newcastle disease virus in chickens by titration of antibodies in egg yolk.

Attempts were made to establish methods for indirect prediction of hemagglutination inhibition (HI) antibody titers to Newcastle disease virus (NDV) in sera of laying hens and day-old chicks by determining if these are correlated to HI titers in egg yolks. For this purpose, geometric means of HI antibody titers in sera from 60 hens, yolks from 60 matched eggs, and sera from 180 day-old chicks of an identical vaccination program were measured and plotted. There was a significant correlation between HI antibody titers in yolks (X) and hens (Y), with a linear regression of Y = 23.24 + 0.47X and a correlation coefficient of r = 0.65. The linear regression between HI antibody titers in yolks (X) and chicks (Y) was Y = 6.33 + 0.36X (r = 0.58). Immunity to NDV in hens and their offspring can be maintained effectively, and the proper time for the vaccination or booster can be determined by reference to HI titers predicted from the linear regression in the present study. The approach of testing egg yolk for HI titers provides a feasible alternative to determining HI titers from blood samples and eliminates stress in birds during blood sampling.     

Vaccination of 1-day-old chicks with fowlpox virus by the aerosol, drinking water, or cutaneous routes

Administration of 10(4) mean cell-culture infectious dose (CCID50) per ml of a plaque-purified derivative of a commercial fowlpox virus (FPV) vaccine to 1-day-old chicks by aerosol or drinking water gave inconsistent serological responses and little evidence of protective immunity. In contrast, cutaneous vaccination with the same preparation protected against challenge with virulent FPV at 4 weeks of age. Administration of the vaccine at a concentration of 10(6) CCID50 per ml by the drinking-water route was as effective as conventional cutaneous vaccination in terms of the serological response in an enzyme-linked immuno-sorbent assay and in terms of protection against challenge. Drinking-water vaccination at 2 days of age was no more effective than vaccination on day 1, and oral dosing with the vaccine was less effective than incorporation of the vaccine in the drinking water. It was concluded that 1-day-old chicks may be vaccinated against fowlpox by the drinking-water route if the vaccine contains a sufficiently high concentration of virus.     

Vaccination against Newcastle disease with a recombinant baculovirus hemagglutinin-neuraminidase subunit vaccine

Vaccination of chickens with an oil-emulsion vaccine containing a recombinant baculovirus that expressed the hemagglutinin-neuraminidase (HN) of Newcastle disease virus (NDV)-induced hemagglutination-inhibition (HI) and virus-neutralizing antibodies against NDV. HI antibody titers obtained in response to vaccination with the live recombinant virus were higher than those obtained when the recombinant was inactivated with beta-propiolactone, and the titers were lower than those obtained in response to the same HN concentrations in live or beta-propiolactone-inactivated NDV strain B1. The serological response to the recombinant baculovirus was differentiated from the response to NDV by an enzyme-linked immunosorbent assay in which purified NDV nucleoprotein was used as antigen. Chickens vaccinated with the live recombinant or with inactivated NDV resisted an oculonasal challenge with the neurotropic velogenic Texas GB strain of NDV, which was lethal in unvaccinated controls. It was concluded that the HN protein of NDV expressed as a subunit by a recombinant baculovirus was protective against Newcastle disease.