Diagnosis of Canine Brucellosis: Comparison between Serological and Microbiological Tests and a PCR Based on Primers to 16S-23S rDNA Interspacer

A pair of primers directed to 16S-23S rDNA interspacer (ITS) was designed directed to Brucella genetic sequences in order to develop a polymerase chain reaction (PCR) putatively capable of amplifying DNA from any Brucella species. Nucleic acid extracts from whole-blood from naive dogs were spiked with decreasing amounts of Brucella canis RM6/66 DNA and the resulting solutions were tested by PCR. In addition, the ability of PCR to amplify Brucella spp. genetic sequences from naturally infected dogs was evaluated using 210 whole-blood samples of dogs from 19 kennels. The whole-blood samples collected were subjected to blood culture and PCR. Serodiagnosis was performed using the rapid slide agglutination test with and without 2-mercaptoethanol. The DNA from whole blood was extracted using proteinase-K, sodium dodecyl sulphate and cetyl trimethyl ammonium bromide followed by phenol–chloroform purification. The PCR was capable of detecting as little as 3.8 fg of Brucella DNA mixed with 450 ng of host DNA. Theoretically, 3.8 fg of Brucella DNA represents the total genomic mass of fewer than two bacterial cells. The PCR diagnostic sensitivity and specificity were 100%. From the results observed in the present study, we conclude that PCR could be used as confirmatory test for diagnosis of B. canis infection.     

Adenocarcinoma of the parotid salivary gland in a cow

A 6‐year‐old Girolando dairy cow was presented for evaluation of a large subcutaneous facial mass. Fine‐needle aspirates of the mass contained many neoplastic cells with high nuclear:cytoplasmic ratios arranged in sheets and loosely cohesive clusters with streaming erythrocytes and neutrophils in the background. Neoplastic cells were 13–25 μm in diameter and were round to cuboidal with variably distinct borders. Based on the signalment, anatomic location, and cytologic findings, differential diagnoses included salivary adenocarcinoma, squamous cell carcinoma, and mucoepidermoid carcinoma. The cow was euthanized and a necropsy was performed. The primary neoplasm arose from the left parotid salivary gland and meastatic tumor was found in the regional lymph nodes and lung. Histologically, the tumor was composed of anastomosing and irregular solid islets surrounded by scant stroma. Cells were negative for periodic acid‐Schiff (PAS), PAS‐diastase, and Alcian blue pH 2.5 stains, used to detect mucin. On immunohistochemical analysis, neoplastic luminal salivary gland cells expressed cytokeratin, but not S100, α‐smooth muscle actin, or vimentin. Peripheral cells of neoplastic islets were immunoreactive for p63. The final diagnosis was nonsecretory adenocarcinoma of the parotid salivary gland.     

Shade tolerance, photoinhibition sensitivity and phenotypic plasticity of Ilex aquifolium in continental Mediterranean sites

Shade tolerance, plastic phenotypic response to light and sensitivity to photoinhibition were studied in holly (Ilex aquifolium L.) seedlings transported from the field to a greenhouse and in adult trees in the field. All plants were growing in, or originated from, continental Mediterranean sites in central Spain. Seedlings tolerated moderate but not deep shade. Mortality was high and growth reduced in 1% sunlight. Survival was maximal in 12% sunlight and minimal in full sunlight, although the relative growth rate of the seedlings surviving in high light was similar to that of plants in moderate shade. Maximum photochemical efficiency at predawn was significantly lower in sun plants than in shade plants in the field, revealing chronic photoinhibition that was most pronounced in winter. Plasticity in response to available light varied according to the variable studied, being low for photosynthetic capacity and stomatal conductance, and high for specific leaf area, root:shoot ratio and leaf area ratio, particularly in seedlings. Differences in water relations and hydraulic features between sun and shade plants in the field were marginal. High water potential at the turgor loss point of field-grown plants suggested that holly is sensitive to drought during both the seedling and the adult stage. Low relative growth rates in both high and low light with low physiological plasticity in response to light indicate the existence of a stress-tolerance mechanism. We conclude that holly is a facultative understory plant in areas of oceanic and relatively mild climate, but an obligate understory plant in dry continental areas such as the study site. The impact of abandonment of traditional management practices and climate change on these Mediterranean populations is discussed.     

Technical Note: In vivo estimation of lipogenesis using a bolus injection of [U-13C]glucose in pigs

The use of radioactive isotopes to measure de novo lipogenesis in pigs has been well established. Different from radioactive isotopes, stable isotopes present little or no risk to human and animal subjects. Therefore, the objective of this study was to adapt the method of bolus injection of radioactive glucose (¹⁴C) to use ¹³C-labeled glucose to estimate de novo lipogenesis in finishing pigs. Five vein-catheterized gilts received 3.0 kg/d of a commercial diet for 2 wk. On the last day, the pigs received a bolus injection of [U-¹³C]glucose (12 mg/kg body weight). A serial of blood samples was taken for 4 h to determine the glucose rate of disappearance (R_d) from plasma glucose isotopic enrichment (IE). The ¹³C IE of lipids was determined from adipose tissue biopsies collected at 1, 2, and 3 h after the bolus injection and from adipose tissue collected after pig euthanasia 4 h after the bolus. Lipogenesis was estimated from the incorporation of ¹³C from glucose into adipose tissue lipids. Glucose R_d, estimated using a double-exponential function, averaged 5.4 ± 1.4 mmol/min. The IE of lipids increased linearly during the 4 h following the bolus injection (P < 0.05). The rate of incorporation of glucose into lipids, estimating lipogenesis, averaged 9.0 µg glucose/(min × g of lipids) 4 h after the bolus injection. In conclusion, the in vivo method using a bolus injection of [U-¹³C]glucose allows a successful estimation of de novo lipogenesis in finishing pigs.     

Evidence for two gene pools of the Lima bean,Phaseolus lunatus L., in the Americas

Summary The lima bean,Phaseolus lunatus L., is a bean species with a broad distribution in the Americas that rivals that of common bean (P. vulgaris). In order to better understand the organization of genetic diversity and the pattern of domestication in lima bean, a review was conducted of the available information on the geographic distribution of wild and cultivated forms of this species. In addition, one-dimensional SDS polyacrylamide gel electrophoresis of seed proteins was also conducted on a sample of 84 wild, 6 weedy, and 426 cultivated forms. Results show that wild forms can be divided into two groups, one with smaller seeds and a very extensive distribution that includes Mexico, Central America, and the eastern slope of the Andes, and the other with a more circumscribed distribution on the western slope of the Andes in Ecuador and northern Peru. Electrophoretic analyses of seed proteins confirmed this subdivision and, additionally, showed that the large-seeded cultivars had been domesticated from the large-seeded wild lima beans in western South America. For the small-seeded lima bean cultivars, it was not possible to determine a domestication center as the most abundant protein pattern in the cultivars also had a widespread distribution in the small-seeded wild progenitor. Electrophoretic analyses showed, however, that domestication led to a reduction of genetic diversity in the small-seeded, Mesoamerican group, but not in the large-seeded group. The latter may be due to insufficient sampling of the larger-seeded, wild germplasm.     

Reactor technique for prediction of dissolved oxygen profiles in streams

A laboratory experiment that can provide significant design information to an environmental scientist or engineer can be performed rapidly and inexpensively with an open stirred-reactor that simulates a receiving stream. The experiment requires only a laboratory beaker, stirring apparatus, dissolved oxygen meter, wastewater sample and receiving stream sample. Wastewater and receiving stream water are mixed in the laboratory beaker and are stirred at different speeds to obtain different reaeration rates. A probe is used to monitor dissolved oxygen concentrations of the mixture. A series of experiments were performed at different waste loadings and different reaeration rates. Sample results are shown which indicate the depletion of dissolved oxygen due both to carbonaceous and nitrogeneous oxygen demands. This technique can be used to predict response of a receiving stream to a waste discharge without use of a mathematical model. Results are shown which illustrate the difference between experimental results and theoretical predictions.     

Anti-Phytophthora cinnamomi activity of Phlomis purpurea plant and root extracts

Phlomis purpurea (Lamiaceae), found in Quercus suber and Quercus ilex ssp. rotundifolia forest habitats in southern Portugal, is a non-host for the oomycete Phytophthora cinnamomi, the main biotic factor involved in cork oak and holm oak decline in the Iberian Peninsula. The effect of P. purpurea crude ethanol root extract was evaluated in vitro on P. cinnamomi mycelial growth, sporangial production, zoospore release and germination as well as on chlamydospore production and viability. The protection of cork oak against infection by the pathogen was also evaluated in planta. At 10 mg ml^?1, in vitro inhibition of the pathogen structures was 85–100 %. In addition, P. purpurea plants were shown to protect Q. suber and Q. ilex from P. cinnamomi infection and to reduce the inoculum potential in glasshouse trials, indicating the ability to reduce root infection by the pathogen. The results suggest that P. purpurea has the potential to reduce disease spread and that their root extracts could provide candidate substances for control of the important pathogen, P. cinnamomi.     

Restricting inbreeding while maintaining selection response for weight gain in Mus musculus

An experiment with mice was designed to test the relative efficiency of three selection methods that help to minimize the rate of inbreeding during selection. A common house mice (Mus musculus) population was selected for 17 generations to increase the weight gain between 21 and 42 days. The population was split at random into three lines A, B and C where three selection methods were applied: individual selection and random mating, weighted selection with random mating and individual selection with minimum coancestry mating, respectively. There were three replicates for each line. Cumulated selection response was similar in the three lines, but there were differences in the level of inbreeding attained (in percentage): 31.24 (method A), 24.72 (method B) and 27.88 (method C). As consequence, lines B and C (weighted selection and minimum coancestry) showed a lower value of deterioration of fitness traits (the intrauterine mortality and the mortality at birth) than line A (random mating).