Immunolabelling of Atrazine Residues in Soil

An immunolabelling procedure was developed to detect bound pesticide residues in soil using atrazine as an example. Polyclonal antibodies directed against atrazine were raised in rabbits. Antigen-binding fragments (F_ab) were prepared and coupled to the fluorescent stain Rose Bengal B. The dye conjugate produced a fluorescence signal which was related to the amount of atrazine found in native soil samples. Preincubation with unlabelled immunoglobulines (IgG) from preimmune sera proved to be essential for blocking unspecific binding of the antibodies which varied greatly between different samples of soil material. Labelled antibodies, whose specific binding sites had been blocked by free atrazine, were used as controls. Strong unspecific labelling, which also could be suppressed with IgG from preimmune sera, was observed in a soil under a barn. Changes due to the permanent dryness and the lack of aeration are discussed as possible causes.     

A real-time polymerase chain reaction assay for quantification of Edwardsiella ictaluri in catfish pond water and genetic homogeneity of diagnostic case isolates from Mississippi

A quantitative polymerase chain reaction (qPCR) assay was developed for the detection and quantification of Edwardsiella ictaluri in channel catfish Ictalurus punctatus pond water using modifications to a published E. ictaluri-specific qPCR assay and previously established protocols for the molecular detection of myxozoan parasites in catfish ponds. Genomic DNA equivalents indicative of the number of bacteria in a sample were determined and standard curves correlating to bacterial numbers were established. The assay was found to be highly repeatable and reproducible, with a linear dynamic range of five orders of magnitude. There was no interference of the assay from the presence of large quantities of nontarget DNA. Known quantities of bacteria were added to sample volumes of 40 or 500 mL of pond water collected from several different ponds. The minimum level of detection was approximately 100 cell equivalents (CE) in 40 (2.5 CE/mL) or 500 mL of pond water (0.2 CE/mL). Sample volumes of 40 mL yielded the most consistent results, which were not significantly different from those obtained from broth culture alone. Cell equivalents determined by qPCR in 40-mL pond water samples spiked with known quantities of bacteria were within one order of magnitude of the actual number of cells added. Repetitive element-based polymerase chain reaction analysis of archived isolates demonstrated the genetic homogeneity of E. ictaluri, and consistent amplification of these isolates by qPCR analysis demonstrated the stability of the PCR target. The assay described here provides a reliable method for the detection and quantification of E. ictaluri in pond water and will be an invaluable tool in epidemiological studies. Additionally, the assay provides a way to evaluate the effects that vaccination, antibiotic treatments, and restricted feeding practices have on E. ictaluri populations during an outbreak. Information obtained with these tools will aid in optimizing disease management practices designed to maximize productivity while minimizing losses.     

Oral Vaccination of Channel Catfish against Enteric Septicemia of Catfish Using a Live Attenuated Edwardsiella ictaluri Isolate

Enteric septicemia of catfish (ESC), caused by Edwardsiella ictaluri, is the most problematic bacterial disease affecting catfish aquaculture in the southeastern United States. Efforts to develop an effective ESC vaccine have had limited industrial success. In commercial settings, ESC vaccines are typically administered by immersion when fry are transferred from the hatchery to rearing ponds. While this approach is a practical method of mass delivery, this strategy administers vaccines to very young fish, which lack a fully developed immune system. To circumvent this limitation, an oral vaccination strategy was evaluated as a means of immunizing catfish at the fingerling stage of production, when fish possess a more complete immune arsenal. A virulent E. ictaluri isolate (S97-773) was attenuated by successive passage on media containing increasing concentrations of rifamycin. In laboratory trials, cultured vaccine was diluted and mixed with feed (100 mL diluted vaccine/454 g feed). This mixture was then fed to Channel Catfish Ictalurus punctatus fingerlings. Two separate dilutions of cultured vaccine (1:10 and 1:100) were used to create the vaccine–feed mixture, equating to estimated doses of 5 × 10⁷ and 5 × 10⁶ CFU/g of feed, respectively. After 30 d, catfish were exposed by immersion (1 × 10⁶ CFU/mL) to the virulent parental strain of E. ictaluri. The target dose (1:100 dilution, ～5 × 10⁶ CFU/g of feed) offered exceptional protection (relative percent survival = 82.6–100%). In addition, negligible deaths occurred in fish vaccinated at 10 times the target dose (1:10 dilution, ～5 × 10⁷ CFU/g of feed). In pond trials, antibody production increased 18-fold in orally vaccinated fish. When compared with nonvaccinated controls, vaccination significantly improved survival, feed fed, feed conversion, biomass produced, and total harvest. This research demonstrates Channel Catfish can be successfully immunized in a commercial setting against E. ictaluri with a single dose of an orally delivered, live attenuated, E. ictaluri vaccine.

Received July 31, 2014; accepted March 2, 2015     

Wolbachia establishment and invasion in an Aedes aegypti laboratory population

A proposed strategy to aid in controlling the growing burden of vector-borne disease is population replacement, in which a natural vector population is replaced by a population with a reduced capacity for disease transmission. An important component of such a strategy is the drive system, which serves to spread a desired genotype into the targeted field population. Endosymbiotic Wolbachia bacteria are potential transgene drivers, but infections do not naturally occur in some important mosquito vectors, notably Aedes aegypti. In this work, stable infections of wAlbB Wolbachia were established in A. aegypti and caused high rates of cytoplasmic incompatibility (that is, elimination of egg hatch). Laboratory cage tests demonstrated the ability of wAlbB to spread into an A. aegypti population after seeding of an uninfected population with infected females, reaching infection fixation within seven generations.     

The speciation of some tributyltin compounds using Mössbauer spectroscopy in different estuarine sediments

The speciation of several tributyltin (TBT) compounds used in marine antifouling paints was studied by tin Mössbauer spectroscopy in both spiked anoxic and oxic estuarine sediments. These compounds included TBT acetate, chloride, fluoride, and oxide. To assist in the determination of the products, possible speciation products were also examined: TBT carbonate, sulfate, and sulfide. The study indicated that TBTF remains in its polymeric form after interaction with both types of sediments. TBT sulfate is unaltered in the spiked sediments. The data reported in this paper support the hypothesis that the speciation of the other TBT compounds examined in spiked sediments depends upon the nature of the sediment.     

Histological and computed tomographic evaluation of a parasitic conjoined twin in hybrid catfish (Ictalurus punctatus [Rafinesque] X Ictalurus furcatus [Lesueur])

There is growing use of hybrid catfish (Ictalurus punctatus ♀ X Ictalurus furcatus ♂) in commercial aquaculture to utilize hybrid vigour to improve production A conjoined twin specimen found during the course of production studies by the United States Department of Agriculture Catfish Genetic Research Unit (USDA‐CGRU) was submitted to the Aquatic Research and Diagnostic Laboratory (ARDL). After preliminary inspection, it was transported to Mississippi State University, College of Veterinary Medicine for further evaluation. The specimen was examined using both computed radiography and computed tomography antemortem. Following humane euthanasia, the specimen was examined both grossly and histologically. Tissues from both fish were also submitted for genetic analysis to determine whether twins were derived from the same egg. This report records the presentation and examination of a pair of conjoined hybrid catfish (I. punctatus X Ictalurus furcatus).     

In vivo measurement of flatulence and nutrient digestibility in dogs fed poultry by-product meal, conventional soybean meal, and low-oligosaccharide low-phytate soybean meal

OBJECTIVE: To determine an optimal window for determining peak flatulence and evaluate the effects of oligosaccharides and supplemental beta-mannanase in soybean meal-based diets on nutrient availability and flatulence. ANIMALS: 6 dogs. PROCEDURES: Dogs were used in a 2 x 3 factorial arrangement of treatments in a 6 x 6 Latin square experiment to evaluate the digestibility, flatulence, and fecal odor metabolites of low-oligosaccharide low-phytate soybean meal (LLM), conventional soybean meal (SBM), and poultry by-product (PBP) meal diets with or without supplemental beta-mannanase (5 g/kg). RESULTS: Enzyme supplementation had no effect on total tract dry matter (DM), nitrogen digestibility, or digestible energy; however, differences between protein sources did exist for total tract DM digestibility and digestible energy. The PBP meal had higher DM digestibility and digestible energy (mean, 0.913 and 4,255 cal/g), compared with soy-based diets (mean, 0.870 and 4,049 cal/g). No differences were detected for any treatment regardless of protein source or addition of supplemental enzyme for any flatulence components analyzed. No differences were detected for all fecal odor metabolites regardless of addition of supplemental enzyme; however, differences between protein sources were detected. The PBP meal had lower concentrations of carboxylic acids and esters and higher concentrations of heterocycles, phenols, thio and sulfides, ketones, alcohols, and indoles than LLM and SBM. CONCLUSIONS AND CLINICAL RELEVANCE: Diets containing < 22.4 g of stachyose/kg and < 2 g of raffinose/kg did not alter digestibility or increase flatulence in dogs.