TaqMan real-time polymerase chain reaction assay for rapid detection of Flavobacterium columnare

Flavobacterium columnare is a ubiquitous Gram-negative bacterium that causes columnaris disease in a wide variety of fish worldwide. Timely detection of this bacterium is important to prevent its spreading and to reduce the economic loss to fish farmers. We developed a TaqMan-based real-time polymerase chain reaction (PCR) targeting a 113 bp nucleotide region of the chondroitin AC lyase gene of F. columnare G₄. Specificity of the assay evaluated with 20 isolates of F. columnare and 15 other taxonomically or ecologically related bacteria revealed that the primers and probe were 100% specific for detection of F. columnare. The sensitivity limit of detection of F. columnare in pure cultures, over a range of dilutions [3.1 × 10⁰–3.1 × 10⁶ colony-forming units (CFU) mL⁻¹], was observed to be ∼3 bacterial cells. The lowest limit of detection in nucleic acids from pure culture of F. columnare was 5.4 fg and the assay was linear with the log of amount of nucleic acid (R²=0.994) over that range (5.4 ng–5.4 fg). In tissues (blood, gills and kidney) of F. columnare experimentally infected fish, the bacterial numbers measured by TaqMan real-time PCR ranged from 3.4 × 10⁰ to 9.5 × 10⁵ CFU mL⁻¹. In both F. columnare experimentally infected and spiked samples, positive PCR results were confirmed by bacteriological culture with 100% agreement. The TaqMan real-time PCR developed in this study is specific, sensitive and reproducible for the detection and quantitation of F. columnare in infected fish.     

Estimating uncertainty in fish stock assessment and forecasting

A variety of tools are available to quantify uncertainty in age‐structured fish stock assessments and in management forecasts. These tools are based on particular choices for the underlying population dynamics model, the aspects of the assessment considered uncertain, and the approach for assessing uncertainty (Bayes, frequentist or likelihood). The current state of the art is advancing rapidly as a consequence of the availability of increased computational power, but there remains little consistency in the choices made for assessments and forecasts. This can be explained by several factors including the specifics of the species under consideration, the purpose for which the analysis is conducted and the institutional framework within which the methods are developed and used, including the availability and customary usage of software tools. Little testing of either the methods or their assumptions has yet been done. Thus, it is not possible to argue either that the methods perform well or perform poorly or that any particular conditioning choices are more appropriate in general terms than others. Despite much recent progress, fisheries science has yet to identify a means for identifying appropriate conditioning choices such that the probability distributions which are calculated for management purposes do adequately represent the probabilities of eventual real outcomes. Therefore, we conclude that increased focus should be placed on testing and carefully examining the choices made when conducting these analyses, and that more attention must be given to examining the sensitivity to alternative assumptions and model structures. Provision of advice concerning uncertainty in stock assessments should include consideration of such sensitivities, and should use model‐averaging methods, decision tables or management procedure simulations in cases where advice is strongly sensitive to model assumptions.     

Global phylogeography of the smooth hammerhead shark: Glacial refugia and historical migration patterns

1. Extreme climate changes during the Cenozoic Era strengthened different biogeographical barriers that decreased the connectivity among populations, triggering lineage diversification of different species worldwide.
2. The mitochondrial DNA control region was employed to explore the phylogeography of Sphyrna zygaena, a globally distributed species threatened by unsustainable, illegal, unreported and unregulated fisheries triggered by the international shark fin trade. It is listed as ‘Vulnerable’ by the IUCN Red List and its trade is regulated by CITES Appendix II.
3. Only 13 haplotypes were found with low genetic diversity levels (hd = 0.686 ± 0.014; π = 0.00206 ± 0.00004) compared with other species of the Sphyrnidae family. The species has a very strong phylogeographic population structure among the Atlantic, Indian and Pacific Oceans (Φ_ST = 0.79132). Worldwide, there are six distinct populations with some haplotype sharing.
4. These populations are probably connected by a stepping-stone dispersal of a small number of migrants per generation from the Indo-Pacific towards the Atlantic. Modelling suggests that S. zygaena diverged into two lineages around 6.96 million years ago which have been isolated in glacial refuges in the Atlantic and Indo-Pacific oceans; and after deglaciation, a population expansion probably permitted secondary contact.
5. Conservation plans to establish differentiated management units should be adopted in each of the identified populations. Among these, the Eastern Central Atlantic and West Indo-Pacific are the most important areas for the species considering the historical migration routes that act as a bridge connecting the Atlantic and Indo-Pacific Oceans while the Gulf of Guinea connects the Atlantic populations. Still, further studies are required to know if these populations are also linked with nursery areas for the species.
6. The results herein can help to delimit the main evolutionarily significant units to implement effective policies to establish differentiated management units as starting points to genetic monitoring programmes for Sphyrna zygaena.

     

Additional notes to the cheklist of cuban cultivated plants (1)

Summary New information is presented for the checklistof Cuban cultivated plants (Esquivel et al. 1989). 79 new species were included. For another 50 species new items are indicated.

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Ergänzende Notizen zur Liste der kubanischen Kulturpflanzen (1)

Zusammenfassung Neue Informationen zur Liste der kubanischen Kulturpflanzen (Esquivel et al. 1989) werden gegeben. 79 Arten wurden neu aufgenommen. Für weitere 50 Arten werden ergänzende Angaben gebracht.

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(I)

(Esquivel et al. 1989). 79 . 50 .

     

Microbial community composition on native and drastically disturbed serpentine soils

During construction of roads, entire hillsides can be cut away, dramatically disturbing the ecosystem. Microbial communities play important, but poorly understood roles in revegetating roadcuts because of the many functions they perform in nutrient cycling, plant symbioses, decomposition, and other ecosystem processes. Our objective was to determine relationships among microbial community composition, soil chemistry, and disturbance on a serpentine soil disturbed by a roadcut and then partially revegetated. We hypothesized that the adjacent undisturbed serpentine soil would have a different microbial community composition from barren and revegetated sections of the roadcut and that undisturbed soils would have the greatest microbial biomass and diversity. We measured phospholipid fatty acids (PLFA) and soil nutrient concentrations on barren and revegetated sections of the roadcut and on adjacent undisturbed serpentine and nonserpentine soils. Most roadcut samples had soil chemistry similar to the serpentine reference soil. The microbial biomass and diversity of barren sites was lower than that of revegetated or the serpentine reference site. The nonserpentine reference site had significantly (P≤0.05) greater microbial biomass than serpentine or disturbed sites but significantly lower relative proportions of actinomycetes, and slow growth biomarkers. The Barren site had the lowest microbial biomass and a significantly (P≤0.05) greater proportion of that biomass was fungi. Barren, revegetated, and serpentine sites all had dissimilar microbial community composition. The composition of the revegetated communities, however, was intermediate between the serpentine reference and barren soils, suggesting that community composition of revegetated soils is approaching that of an undisturbed site with similar soil chemistry.     

Characterization and Degradation of Petroleum Hydrocarbons Following an Oil Spill into a Coastal Environment of South Texas, U.S.A.

Petroleum hydrocarbons were characterized at eleven sites withinthe sediments of a coastal stream in south Texas, U.S.A. following a medium sized crude oil spill. Bank and open-water(deep) sediments were collected at each site. Hydrocarbonstargeted for analysis included 22 aliphatic hydrocarbons (C-11to C-34) and 16 polynuclear aromatic hydrocarbons. Sedimentconcentrations were measured at intervals of 1, 2, 3, 4, 6, and12 months post-spill. Higher hydrocarbon concentrations were observed for a longerduration within the deep sediments than bank sediments. Initialhydrocarbon constituents in impacted sediments matched the crudeoil fingerprint accurately with the exception of the lighter-endhydrocarbons. The lighter-end aliphatic hydrocarbons areaffected immediately by evaporation and dissolution processesduring the spill event and were found below the detection levelsat most of the sites. Total hydrocarbon concentrations insediments within each hydrocarbon group returned to backgroundlevels by the end of the study period. Observed decreases inhigh molecular weight polycyclic aromatic hydrocarbons (PAH)concentrations exceeded known environmental degradation rateswhich suggests the influence of a sediment transport process. Overall, the fate of petroleum hydrocarbons within this type ofenvironment were likely related to both degradation and sedimenttransport processes. By the end of the study period, most individual PAH constituent concentrations were below thresholdconcentrations thought to produce toxic effects in marine andestuarine organisms. PAH constituents concentrations remainingabove threshold concentrations included benz(a)anthracene,chrysene, and benzo(a)pyrene.     

Milk excretion of ivermectin and moxidectin in dairy sheep: assessment of drug residues during cheese elaboration and ripening period

Ivermectin (IVM) and moxidectin (MXD) are broad-spectrum endectocide antiparasitic drugs extensively used in food-producing animals. The patterns of IVM and MXD excretion in milk were comparatively characterized following their subcutaneous administration (200 microg.kg(-1) of body weight) to lactating dairy sheep. The relationship between milk excretion and plasma disposition kinetics of both compounds was characterized. A pool of milk collected from all of the animals in each experimental group was used for cheese elaboration. IVM and MXD residual concentrations were assessed during the cheese-making process and ripening period. IVM and MXD concentrations were measured in plasma, milk, and milk product (whey, curd, and cheese) samples using an HPLC-based methodology with fluorescence detection. IVM and MXD were extensively distributed from the bloodstream to the mammary gland, and large quantities, particularly of MXD, were excreted in milk. Residual concentrations of both compounds were recovered in milk up to 30 (IVM) and 35 (MXD) days post-treatment. The total fraction of the administered dose excreted in milk for MXD was significantly higher than that of IVM. During cheese production, the highest residual concentrations of both molecules were measured in the curd. Thirty-four percent of the total drug residue measured in the pooled milk collected from treated sheep was lost during the cheese-making process. The lowest residual concentrations were measured in the whey. IVM and MXD concentrations in the elaborated cheese tended to increase during the ripening period, reaching the highest residual level at 40 days of cheese maturation. The long persistence of milk residual concentrations of MXD and IVM in lactating dairy sheep and the high concentrations found in cheese and other milk-related products should be seriously considered before recommendation of the extralabel use of these antiparasitic drugs in dairy animals.     

Seasonal distribution of air-borne protozoa in Mexico City and its suburbs

A survey was carried out over a 1-yr period to isolate protozoa suspended in the air of Mexico City and its suburbs. Sampling stations were placed at four cardinal points of the metropolitan area. Selective media were used to culture the protozoa isolated. Specialized taxonomic keys were used for identification and a statistical analysis was performed to determine the correlations between physicochemical and biological parameters. 63 strains were isolated. Species of Kinetoplastida and Chrysomonadida were most abundant, namely species of the genera Bodo, Cercobodo, Monas and Helkesimastix. Amoeboflagellates found included Mastigamoeba and Tetramitus. The only ciliate isolated was Colpoda steini. The greatest number of strains was yielded by Bodo repens and Cercobodo radiatus. Several times during the period of the study SO₂, O₃, CO, NO, and NO₂ exceeded the permissible levels established by the Mexican government. From the results of this study it was concluded that the ability of protozoa to form cysts and cyst size were important factors for their presence, survival, abundance and diversity in the atmosphere. The main source of air-borne protozoa was the soil. Factors that favored the incidence and diversity of the isolates were wind speed and direction, high relative humidity, generation of frequent dust-storms, resuspension of protozoa by vehicular traffic, proximity to garbage dumps and large extensions of bare soil, and temperature (the latter only when it did not favor the production of high levels of O₃). Factors that were hostile to the aerial protozoa were industry and their contaminants, and high concentrations of O₃ and SO₂, especially in winter time when thermal inversions occurred. Soil cover was also associated with a reduction in the incidence and diversity of the aerial protozoa. This study demonstrates that there are viable cysts of protozoa in the atmosphere of Mexico City, that may have potential importance in the case of certain kinds of human allergies and diseases. Further research is needed to find out the aerial presence of viable cysts of obligatory, amphizoic or opportunistic protozoan parasites, and to clarify the qualitative and quantitative effects of the local meteorological and physico-chemical environment on the free-living protozoa present in the atmosphere.