Transformation of Anaplasma marginale

The tick-borne pathogen, Anaplasma marginale, has a complex life cycle involving ruminants and ixodid ticks. It causes bovine anaplasmosis, a disease with significant economic impact on cattle farming worldwide. The obligate intracellular growth requirement of the bacteria poses a challenging obstacle to their genetic manipulation, a problem shared with other prokaryotes in the genera Anaplasma, Ehrlichia, and Rickettsia. Following our successful transformation of the human anaplasmosis agent, A. phagocytophilum, we produced plasmid constructs (a transposon bearing plasmid, pHimarAm-trTurboGFP-SS, and a transposase expression plasmid, pET28Am-trA7) designed to mediate random insertion of the TurboGFP and spectinomycin/streptomycin resistance genes by the Himar1 allele A7 into the A. marginale chromosome. In these trans constructs, expression of the fluorescent and the selectable markers on the transposon, and expression of the transposase are under control of the A. marginale tr promoter. Constructs were co-electroporated into A. marginale St. Maries purified from tick cell culture, and bacteria incubated for 2 months under selection with a combination of spectinomycin and streptomycin. At that time, ≤1% of tick cells contained colonies of brightly fluorescent Anaplasma, which eventually increased to infect about 80–90% of the cells. Cloning of the insertion site in E. coli and DNA sequence analyses demonstrated insertion of the entire plasmid pHimarAm-trTurboGFP-SS encoding the transposon in frame into the native tr region of A. marginale in an apparent single homologous crossover event not mediated by the transposase. Transformants are fastidious and require longer subculture intervals than wild type A. marginale. This result suggests that A. marginale, as well as possibly other species of Anaplasma and Ehrlichia, can be transformed using a strategy of homologous recombination.     

Fossil evidence for evolution of the shape and color of penguin feathers

Penguin feathers are highly modified in form and function, but there have been no fossils to inform their evolution. A giant penguin with feathers was recovered from the late Eocene (~36 million years ago) of Peru. The fossil reveals that key feathering features, including undifferentiated primary wing feathers and broad body contour feather shafts, evolved early in the penguin lineage. Analyses of fossilized color-imparting melanosomes reveal that their dimensions were similar to those of non-penguin avian taxa and that the feathering may have been predominantly gray and reddish-brown. In contrast, the dark black-brown color of extant penguin feathers is generated by large, ellipsoidal melanosomes previously unknown for birds. The nanostructure of penguin feathers was thus modified after earlier macrostructural modifications of feather shape linked to aquatic flight.     

Wild halophytic species as forage sources: Key aspects for plant breeding

The use of wild halophytic species as forage resources in saline environments has gained increasing attention. Argentina ranks third in area of saline soils in the world, with a third of its territory showing various degrees of salinity, sodicity and/or alkalinity. On this type of soils, rangelands are the main forage resource for livestock production. Many wild species have forage potential and can also be used for the rehabilitation of rangelands and for intercropping. Information about these species, as well as on the physiological and genetic bases associated with salinity tolerance, provides relevant tools for efficient selection methods. This study addresses Argentine wild halophyte species with forage potential and describes selection criteria with an emphasis on the following taxa: (a) Poaceae: subfamily Chloridoideae and tribes Paniceae and Triticeae, (b) Fabaceae and (c) Amaranthaceae (formerly known as Chenopodiaceae). The review is intended to contribute to the general discussion on strategies for the improvement of wild plant genetic resources, using forage species naturally growing in saline soils in Argentina as a case study.     

Identification and confirmation of quantitative trait loci for stachyose content in soybean seed

Stachyose is an unfavorable sugar in soybean meal that causes flatulence for non‐ruminant animals. Understanding the genetic control of stachyose in soybean will facilitate the modification of stachyose content at the molecular level. The objective of this study was to identify quantitative trait loci (QTL) associated with seed stachyose content using simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers. A normal stachyose cultivar, ‘Osage’, was crossed with a low stachyose line, V99‐5089, to develop a QTL mapping population. Two parents were screened with 33 SSR and 37 SNP markers randomly distributed on chromosome 10, and 20 SSR and 19 SNP markers surrounding a previously reported stachyose QTL region on chromosome 11. Of these, 5 SSR and 16 SNP markers were used to screen the F_3:4 lines derived from ‘Osage’ x V99‐5089. Seed samples from F_3:5 and F_3:6 lines were analyzed for stachyose content using high‐performance liquid chromatography (HPLC). Composite interval mapping analysis indicated that two stachyose QTL were mapped to chromosome 10 and 11, explaining 11% and 79% of phenotypic variation for stachyose content, respectively. The SSR/SNP markers linked to stachyose QTL could be used in breeding soybean lines with desired stachyose contents. Chi‐square tests further indicated that these two QTL probably represent two independent genes for stachyose content. Therefore, a major QTL was confirmed on chromosome 11 and a novel QTL was found on chromosome 10 for stachyose content.     

Identification of mtDNA lineages of Sus scrofa by multiplex single base extension for the authentication of processed food products

A genetic method to identify the breed of origin could serve as a useful tool for inspecting the authenticity of the increasing number of monobreed foodstuffs, such as those derived from small local European pig breeds. Mitochondrial DNA (mtDNA) is practically the only reliable genomic target for PCR in processed products, and its haploid nature and strict maternal inheritance greatly facilitate genetic analysis. As a result of strategies that sought to improve the production traits of European pigs, most industrial breeds presently show a high frequency of Asian alleles, while the absence or low frequency of such Asian alleles has been observed in small rustic breeds from which highly prized dry-cured and other traditional products are derived. Therefore, the detection of Asian ancestry would indicate nonconformity in Protected Denomination of Origin products. This study presents a single base extension assay based on 15 diagnostic mtDNA single nucleotide polymorphisms to discriminate between Asian and European Sus scrofa lineages. The test was robust, sensitive and accurate in a wide range of processed foodstuffs and allowed accurate detection of pig genetic material and identification of maternal ancestry. A market survey suggested that nonconformity of products derived from Portuguese breeds is an unusual event at present, but regular surveys both in the local populations and in commercial products would be advisible. Taking into consideration the limitations presented by other methodologies, this mtDNA-based test probably attains the highest resolution for the direct genetic test for population of origin in Sus scrofa food products.     

Blood gas analysis and cooximetry in retired racing Greyhounds

Objective – The purposes of this study were to evaluate the oxygen affinity of hemoglobin (Hb) in healthy retired racing Greyhounds via cooximetry, and to establish reference intervals for blood gases and cooximetry in this breed. Design – Prospective clinical study. Setting – University Teaching Hospital. Animals – Fifty‐seven Greyhounds and 30 non‐Greyhound dogs. Interventions – Venous blood samples were collected from the jugular vein and placed into heparinized tubes. The samples were analyzed within 30 minutes of collection using a blood gas analyzer equipped with a cooximeter. Measurements and Main Results – Greyhounds had significantly higher pH, PO₂, oxygen saturation, oxyhemoglobin, total Hb, oxygen content, and oxygen capacity and significantly lower deoxyhemoglobin and P₅₀ when compared with non‐Greyhound dogs. Conclusion – These findings support the fact that this breed is able to carry a higher concentration of total oxygen in the blood. As reported previously, this breed also has lower P₅₀ and, therefore, high oxygen affinity. In light of recent findings suggesting that in certain tissues a high affinity for oxygen is beneficial, this adaptation may be of benefit during strenuous exercise.     

Location and stability of a recombinant ovine prion protein in synthetic humic-like mineral complexes

Location and stability of a recombinant prion protein (recPrP) and its interaction with humic-like complexes were investigated by low-temperature ashing (LTA), thermal gravimetric (TG), and scanning electron microscopy (SEM) analyses. Humic-like complexes were obtained by abiotic polymerization of catechol, one of the possible precursors of soil humic matter, through the catalysis of birnessite, a manganese oxide common in soil environment. The recPrP was immobilized in organomineral complexes via sorption or entrapment. Complexes were treated by LTA, allowing the controlled removal of organic matter layer by layer, from the external to the internal side, with minimal disturbance of mineral constituents. Thermal gravimetric and SEM analyses were performed on specimens before and after LTA treatment. Entrapped recPrP, compared with sorbed, resulted less easily accessible to LTA treatment and showed a higher thermal stability by TGA analyses. On the basis of these findings, we hypothesize that the processes leading to newly formed organic complexes can enhance prion stability in soil and thus influence the environmental diffusion of infectivity.     

Anthocyanin composition of wild Colombian fruits and antioxidant capacity measurement by electron paramagnetic resonance spectroscopy

The qualitative and quantitative anthocyanin composition of four wild tropical fruits from Colombia was studied. Compounds of "mora peque?a" ( Rubus megalococcus Focke.), "uva de árbol" ( Myrciaria aff. cauliflora O. Berg), coral, and motilón ( Hyeronima macrocarpa Mull. Arg.) fruits were separately extracted with methanol-acetic acid (95:5, v/v). The anthocyanin-rich extracts (AREs) were obtained by selective adsorption on Amberlite XAD-7. Each extract was analyzed by HPLC-PDA and HPLC-HRESI-MS(n) with LCMS-IT-TOF equipment in order to characterize the anthocyanin pigments and the coinjection in HPLC using standards allowed identifying the major constituents in each extract. The antioxidant activity was measured by electron paramagnetic resonance (EPR) and UV-vis spectroscopy, using ABTS and DPPH free radicals. The ARE of motilón ( H. macrocarpa Müll. Arg) exhibited the highest radical scavenging activity in comparison to the other extracts. A second-order kinetic model was followed in all of the cases. These results suggested that the studied fruits are promising not only as source of natural pigments but also as antioxidant materials for food industry.