Description of the oöcysts of two new species of Eimeria (Apicomplexa: Eimeriidae) from the rough earth snake Virginia striatula (Serpentes: Colubridae) in Texas and Arkansas

Coccidian oöcysts recovered from the faeces of rough earth snakes Virginia striatula (Serpentes: Colubridae) were found to represent two previously unreported eimerians. Oöcysts of Eimeria desotoensis n. sp. were found in 5/32 (16%) of the snakes and were spherical to ellipsoidal, 18.4 × 17.2 (15–21.5 × 15–19.5) μm, with a thin, single-layered wall; their shape-index (length/width) was 1.07 (1.00–1.23). A micropyle and oöcyst residuum were absent; polar granule were present in 33% of the oöcysts. The sporocysts were ovoidal, 11.5 × 7.6 (10.5–13 × 7–8) μm, with a Stieda body; their shape-index was 1.51 (1.30–1.68). The sporocyst residuum was moderate in size and composed of a cluster of granules. Oöcysts of Eimeria hobartsmithi n. sp. were found in 2/32 (6%) of the snakes and were subspherical to ellipsoidal, 18.0 × 15.7 (16–20 × 15–17) μm, with a thin, single-layered wall; their shape-index was 1.15 (1.02–1.32). A micropyle, oöcyst residuum and polar granule were absent. The sporocysts were elongate, 13.2 × 6.3 (12–14.5 × 6–6.5) μm, with a Stieda body; their shape-index was 2.10 (1.88–2.34). A large sporocyst residuum was present in each sporocyst, often obscuring the sporozoites. In addition to the two new species, oöcysts of E. striatula Upton & McAllister, 1990 were observed in 38% of the snakes.     

Relationship between Enhanced Intensity of Contrast Enhanced Ultrasound and Microvessel Density of Aortic Atherosclerostic Plaque in Rabbit Model

The aim of this study was to evaluate the relationship between enhanced intensity of contrast enhanced ultrasound and microvessel density of aortic atherosclerotic plaque in rabbit model. The abdominal aortas of thirty-six male New Zealand rabbits were damaged by balloon expansion and the animals were then fed a high fat diet for 12 weeks. Twenty-seven plaques on the near aortic wall were detected using conventional ultrasound examination. The maximum thickness of each plaque was recorded. CEUS was performed on these 27 plaques and the time-intensity curves (TICs) were analyzed offline. Using the quantitative ACQ software, features such as the arrival time (AT), time to peak (TTP), baseline intensity (BI), peak intensity (PI) and enhanced intensity (EI) (EI = PI-BI) were assessed. Inter- and intra-observer agreement of EI were assessed using the Bland-Altman test. After CEUS examination, the rabbits were sacrificed for pathological examination and CD34 monoclonal antibody immunohistochemical detection. Microvessel density (MVD) was counted under the microscope. The relationship between indexes of CEUS and the level of MVD was analyzed. There was a good positive linear correlation between EI and MVD (γ = 0. 854, P<0. 001), the intraclass correlations for inter- and intra-observer agreement for EI were 0.73 and 0.82 respectively, suggesting that EI may be act as a useful index for plaque risk stratification in animal models.     

Coccidian Parasites (Apicomplexa: Eimeriidae) from Insectivores: New Species from Shrew Moles (Talpidae) in the United States1

All of 18 shrew moles, Neurotrichus gibbsii, collected in Oregon and Washington were infected with one or more species of coccidia. Three eimerians and one isosporan were identified and described as new species. Sporulated oocysts of Eimeria heterocapita n. sp. were subspheroid to ellipsoid, 25.5 times 21.4 (23–27 times 18–23) μm. A membranous, cap-like structure was present at one pole of the oocyst, but a micropyle, oocyst residuum, and polar body were absent. Ovoid sporocysts were 13.6 times 10.0 (12–15 times 9–11) μm; a compact sporocyst residuum was present, but Stieda, sub-, and parastieda bodies were absent. This species was found in 2 of 18 (11%) hosts. Sporulated oocysts of Eimeria neurotrichi n. sp. were avoid, 17.6 times 13.6 (16–20 times 11–16) μm; micropyle and oocyst residuum were absent, but a polar body was present. Ovoid sporocysts were 10.7 times 5.5 (9–12 times 5–6) μm; Stieda body and sporocyst residuum were present, but sub- and parastieda bodies were absent. This species was found in 2 of 18 (11%) hosts. Sporulated oocysts of Eimeria parastiedica n. sp. were subspheroid, 27.4 times 25.5 (25–30 times 22–28) μm; micropyle, oocyst residuum, and polar body were absent. Ovoid sporocysts, pointed at both ends, were 18.3 times 10.4 (16–20 times 9–11) μm; Stieda, sub-, and parastieda bodies were present as was a sporocyst residuum. This species was found in 2 of 18 (11%) hosts. Sporulated oocysts of Isospora neurotrichi n. sp. were subspheroid, 13.9 times 12.0 (11–16 times 10–15) μm; micropyle and oocyst residuum were absent, but 1–3 polar bodies were present. Ellipsoid sporocysts were 9.2 times 6.1 (8–11 times 5–8) μm; sub- and parastieda bodies were absent, but a Stieda body and sporocyst residuum were present. This species was found in 17 of 18 (94%) hosts. Ten of 18 (56%) hosts were seen to be naturally infected with only one coccidium.     

Culture conditions for production and purification of bioabsorbent from Alcaligenes latus B-16

Glucose and sucrose were favorable carbon sources for the production of bioabsorbent from Alcaligenes latus B-16. Yeast extract at a concentration of 0.5% was also an effective medium for growth. Biopolymers from A. latus B-16 were purified, and the biopolymers were separated into two types of biopolymers: a high-molecular weight component (acid-biopolymer) and a low-molecular weight component (neutral-biopolymer). These two types of biopolymers were shown to be homogeneous on electrophoresis and GPC (Gel Permeation Chromatography on HPLC). The high-molecular weight component was bioabsorbent, and was able to absorb water at more than 1,000 times its own weight. The total yield of acid- and neutral-biopolymers from A. latus B-16 was 20–25 g (dry weight) per liter of culture broth, and they were produced at a ratio of 20:1 (acid:neutral).     

Predation pressure on an imperfect Batesian micicry complex in the presence of alternative prey

Summary Differential predation pressure and the probability of predation on a Batesian mimicry complex and on alternative prey were estimatedin a field experiment. The mimicry complex was composed of a noxious model (Eleodes obscura (Say)) and a palatable mimic (Stenomorpha marginata (LeConte)). House crickets (Acheta domesticus) (Linn.) were used as alternative prey. The experiment was conducted for 23 nights in August and September to approximate the peak seasonal activity time period during which both models and mimics normally are exposed to predation while foraging and depositing eggs. Each night thirty prey in ratios of 16 models: 7 mimics: 7 crickets were exposed for 2.5 h to a suite of predators consisting of pallid bats (Antrozous pallidus), striped skunks (Mephitis mephitis) and ringtails (Bassariscus astutus) that had free access to the prey. The model-mimic ratio was similar to that found in nature. Predators obtained prey on 11 of the 23 nights and preferred the alternative prey (crickets) in proportions higher than was expected from a predation rate that was equal on all species of prey. Mimics were taken by predators at a rate proportional to their abundance, while models were taken at a rate considerably lower than their relative abundance. This suggests that at least some of the predators could distinguish between models and mimics and were willing to eat the mimics at higher frequencies than they were willing to eat the models. However, although the mimicry is not perfect with respect to the entire predator suite, the mimics still gain an advantage by resembling the models, compared to the predation levels on the alternate prey.     

Embryonic development in repeat breeder and virgin heifers seven days after insemination

The aim of this study was to compare the development of embryos from repeat breeder heifers with that of embryos from virgin heifers at 7 days after standing heat. A total of 23 repeat breeder heifers (RBH) and 18 virgin heifers (VH) were utilized. The heifers were between 16 and 30 months of age and most of them were of the Swedish Red and White Breed. Two RBH were heterozygous for the $\text{1}\text{29}$ chromosome translocation, one RBH was a trisomy X and all the other heifers had normal karyotypes. All heifers were inseminated with frozen semen from the same bull and all inseminations were performed by the author. The fertility of the bull was above the average for the AI association to which it belonged. Embryos were collected by a non-surgical technique (89) or after slaughter (19). The morphology of the embryos was examined under a phase-contrast microscope and they were classified as being normal (N), morphologically deviating (MD) or degenerated (D). Thirteen embryos from RBH and 15 from VH were examined for total cell numbers after examination of their morphology.There was no significant difference in recovery rates of embryos between RBH (68%) and VH (76%) but independent of collection method the recovery rate of embryos from VH was numerically higher. The fertilization rate was high in both RBH (89%) and VH (97%). Seventyfour percent of the embryos collected from VH were normal ($\text{23}\text{31}$) while only 28% ($\text{11}\text{40}$) of the embryos collected from RBH had a normal morphology. The difference in number of normal embryos recovered from the two groups of heifers was highly significant (P < 0.005). Exclusion of the RBH heifers with deviating karyotype did not influence this difference. However, there was a tendency to a higher incidence of fertilization failure and morphologically deviating embryos in these heifers. The N embryos had significantly higher total cell numbers (P < 0.005) than the MD embryos but there was no significant difference in total cell numbers between N embryos from RBH or VH.The results of this study strongly indicate a higher incidence of abnormal embryos in RBH than in VH. It is likely that these deviations are followed by an increased incidence of early embryonic death.     

EPR study on the interaction of hexavalent chromium with glutathione or cysteine: Production of pentavalent chromium and its stability

The reduction of hexavalent chromium (Cr(VI)) by glutathione was studied by EPR spectrometry and comparing it with that by cysteine. The characteristics of production of the pentavalent chromium (Cr(V)) species were studied. Two Cr(V) species, which were characterized by g values of 1.995–1.996 and 1.985–1.986, were detected at pH values above 5.0, whereas at pH 3.0 and 4.0 a single species of Cr(V) with a g value of 1.989–1.990 was found. The Cr(V) species were relatively long-lived and most stable at pH 7.0, where signal of two Cr(V) species were observed for more than 30 min. The intensities of the Cr(V) signals were pH-dependent, increasing with an increase in pH from 3.0 to 8.0. At neutral pH, the signal corresponding to the species of the larger g value increased markedly with an increase in glutathione concentration. Stable production of Cr(V) by glutathione was also confirmed by EPR measurements at 77 K. On the other hand, the characteristics of Cr(V) generation by cysteine were quite different. Production of the Cr(V) species was confirmed by a sharp single signal with a g value of 1.984–1.987. The signal intensity corresponding to Cr(V) generation did not change much with a change in pH from 3.0 to 6.0; at pH 7.0 only a small signal was observed, and at pH 8.0 no signal was observed. Moreover, the life-time of the Cr(V) signal was shorter than that observed for the reduction with glutathione. These results suggest that Cr(V) may be stabilized in glutathione solution by its suitable redox potential and ligand structure.     

Structure of Labyrinthula sp. Zoospores*

SYNOPSIS. A species of Labyrinthula closely resembling L. algeriensis was isolated from marsh grass, Spartina alterniflora. Zoosporulation was obtained in ～50% of the cultures, which were grown on a modified Vishniac medium, when yeasts were used as food organisms; when the temperature was ～22 C; when thiamine (0.2 mg/l, biotin (1 μg/l, Ba (1 μg/l, and cholesterol (5 mg/l) were present; and when the cells were grown on a semisolid medium (0.6% agar) rather than higher concentrations of agar. The motile cells were similar to biflagellated fungal zoospores in that they were pyriform and laterally biflagellated. The anterior flagellum (13-15 μ long) possessed what appeared to be 2 rows of mastigonemes and was uniform in diameter while the posterior flagellum (6-10 μ) lacked mastigonemes and was tapered distally into a whiplash. The planonts are apparently not isogametes since planogametic copulation could not be induced and since synaptinemal complexes were not observed in sectioned presporangia.     

Automated determination of tramadol enantiomers in human plasma using solid-phase extraction in combination with chiral liquid chromatography

A sensitive and automated method for the separation and individual determination of tramadol enantiomers in plasma has been developed using solid-phase extraction (SPE) on disposable extraction cartridges (DECs) in combination with chiral liquid chromatography (LC). The SPE operations were performed automatically by means of a sample processor equipped with a robotic arm (ASPEC system). The DEC filled with ethyl silica (50 mg) was first conditioned with methanol and phosphate buffer, pH 7.4 A 1.0-ml volume of plasma was then applied on the DEC. The washing step was performed with the same buffer. The analytes were eluted with 0.15 ml of methanol, and 0.35 ml of phosphate buffer, pH 6.0, containing sodium perchlorate (0.2 M) were added to the extract before injection into the LC system. The enantiomeric separation of tramadol was achieved using a Chiralcel OD-R column containing cellulose tris-(3,5-dimethylphenylcarbamate) as chiral stationary phase. The mobile phase was a mixture of phosphate buffer, pH 6.0, containing sodium perchlorate (0.2 M) and acetonitrile (75:25). The mobile-phase pH and the NaClO₄ concentration were optimized with respect to enantiomeric resolution. The method developed was validated. Recoveries for both enantiomers of tramadol were about 100%. The method was found to be linear in the 2.5–150 ng/ml concentration range [r²=0.999 for (+)- and (−)-tramadol]. The repeatability and intermediate precision at a concentration of 50 ng/ml were 6.5 and 8.7% for (+)-tramadol and 6.1 and 7.6% for (−)-tramadol, respectively.     

Inheritance of photo-control of conidial development in the fungusBipolaris oryzae

The inheritance of light dependence for conidial development inBipolaris oryzae was analyzed using single-ascospore isolates. When a photo-induced strain was crossed with another photo-induced strain, only photo-induced progeny were produced. When a photo-induced strain was crossed with a non-photo-induced (I) strain, photo-induced and nonphoto-induced (I) progeny were produced in a ratio of 1∶1. However, when a photo-induced strain was crossed with a non-photo-induced (II) strain, a non-photo-induced (I) progeny were formed in addition to parental types. It was suggested that genes for photo-control of conidiophore induction and genes for photo-control of conidiophore maturation are located on the same chromosome.     

Correlation of Prostatic Urethral Angle with the Severity of Urinary Symptom and Peak Flow Rate in Men with Small Prostate Volume

Purpose

To evaluate the effects of prostatic anatomical factors on male lower urinary tract symptoms (LUTS) and the peak flow rate (Qmax) in patients with small prostate volume (PV).

Materials and Methods

Records were obtained from a prospectively maintained database of first-visit men with LUTS. Patients whose total PV (TPV) was greater than 30 mL were excluded; 444 patients were enrolled in the study. The TPV, transitional zone volume (TZV), transitional zone index (TZI), intravesical prostatic protrusion (IPP), and prostatic urethral angle (PUA) were measured by transrectal ultrasonography. LUTS were evaluated using the International Prostate Symptom Score (IPSS) and the Overactive Bladder Symptom Score (OABSS) questionnaires. Uroflowmetric measurements were also made.

Results

PUA (r = 0.269, P<0.001), TZV (r = 0.160, P<0.001), and TZI (r = 0.109, P = 0.022) significantly correlated with the IPSS. Qmax (r = −0.334, P<0.001) and OABSS (r = 0.211, P<0.001) correlated only with PUA. In a multivariate regression analysis, PUA and age were independently associated with IPSS, OABSS, and Qmax. For IPSS of 20 or greater, the area under the ROC curve (AUC) of PUA was 0.667 and the cut-off value was 43.7°. When Qmax was 10 mL/s or less, the AUC of PUA was 0.664 and the cut-off value was 43.5°.

Conclusions

PUA has a significant association with symptom severity and Qmax among prostatic anatomical factors analyzed in men with LUTS and small PV. PUA should be considered as an important clinical factor in male LUTS management. Furthermore, the impact of PUA on response to medical treatment and disease progression needs to be investigated.     

Age-specific survivorship analysis ofHeliothis spp. Populations on cotton

Population dynamics of Heliothis virescens (F.) and Heliothis zea (Boddie ) (Lepidoptera: Noctuidae) eggs and larvae were studied for two years in a small plot of cotton, Gossypium hirsutum (L.). Due to morphological and ecological similarities, the pooled Heliothis population was considered for most of the analyses. Two generations of Heliothis eggs and larvae were completed during each year. Stage recruitment was estimated for the eggs and larval instars 2–6, and recruitment variances were estimated by a Monte Carlo method. A modified form of the Weibull distribution was developed and used as a model to characterize survivorship curves for each of the four Heliothis generations. A Type I survivorship curve (mortality rate increasing with age) was inferred for both Generation 1 (early season) data sets, whereas a Type II survivorship curve (mortality rate constant and thus independent of age) was inferred for both Generation 2 (late season) data sets. The shapes of the survivorship curves for the individual H. virescens and H. zea populations were inferred to be the same as those for the pooled populations. Analysis of the contributions of various factors to Heliothis stage-specific mortality indicated that natural enemies (predators and parasites) and the availability of food for larvae were responsible for between-generation differences in survivorship patterns.     

Correlative immunocytochemical and electron microscopic studies: Identification of (Entero)glucagon-somatostatin- and pancreatic polypeptide-like-containing cells in the human colon

Summary Correlative immunocytochemical and electron microscopic studies, using the semi thin-thin technic, were performed to identify the (entero) glucagon, somatostatin and pancreatic polypeptide-like immunoreactive cells of the human colonic mucosa. Mean granule diameter for each cell type was estimated according to two methods and histograms showing the granule size distribution were constructed. A total of 139 immunostained cells identified at the ultrastructural level were analyzed. Mean granule diameter for (entero)glucagon-containing cells was 318±11 nm but a reduction of granule size with age was noteworthy: granules were larger in the fetus (mean diameter 350±15) than in adults (mean diameter 310±10 nm). Somatostatin-containing cells, very rare in adults, were present in the fetal distal colon. Their general mean granule diameter was 354±18 nm but many cells had a mean granule diameter of more than 400 nm. A pancreatic polypeptidelike immunoreactivity was found only in (entero)glucagon-containing cells, pointing out the possible occurrence of both peptides (or of similar sequences) in the same cells. Previous ultrastructural studies dealing with a tentative classification of the human colonic endocrine cells were compared with the present data.This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM).     

Serum Lipoprotein(a) Positively Correlates with Coronary Artery Calcification in Low-Risk Chinese Han Patients: A Study from a Single Center

Background

Elevated plasma levels of lipoprotein(a) (Lp(a)) and a higher degree of coronary artery calcification (CAC) are both considered to be risk factors for atherosclerosis. However, previous studies have demonstrated that the relationship between Lp(a) levels and the degree of CAC indicates significant heterogeneity that may be due to varying ethnicities. The purpose of this study was to examine the predictive power of Lp(a) for CAC as measured by multidetector computed tomography (MDCT) in the Han ethnic group of China.

Methods

A total of 1082 subjects were recruited in this study. The patients were divided into four groups: patients without hypertension or diabetes were group 1, patients with hypertension were group 2, patients with diabetes were group 3 and patients with both hypertension and diabetes were group 4. CAC score (CACs), lipid profiles (Lp(a), LDL, HDL, TG, TC), HbA1C, glucose, personal health history and body morphology were measured in all participants. The predictive power of Lp(a) for calcified atherosclerotic plaque was determined by correlations and ordinal logistic regression.

Results

There was no significant difference in the CACs between group 2 and group 3 (z = 1.790, p = 0.736), and there were significant differences among the other groups. However, there was no significant difference in the total Lp(a) among the 4 groups (χ² = 0.649, p = 0.885). Only In group 1, Lp(a) was a statistically significant predictor of the presence of calcified coronary plaque using ordinal logistic regression.

Conclusions

Levels of Lp(a) positively correlate with CACs among Chinese Han people who are without diabetes and hypertension, suggesting that Lp(a) may be an important risk factor for the presence of calcified atheromas.     

Azotobacter vinelandii biomass production from olive-mill wastewaters for heavy metal recovery

Sixteen litres of olive-mill wastewaters (OMW) diluted to 5% organic matter (d.w.) were inoculated in a fermentor (T=30°C, air FLOW=161min⁻¹, STIRRING=100 rpm) with a strain of Azotobacter vinelandii. After 2 weeks the bacterial biomass was separated by centrifugation and capsular polysaccharide (CPS) and exopolysaccharides were extracted. The apparent molecular weight of CPS was determined by gel filtration. The CPS was entrapped in polyvinyl alcohol membranes which were used to adsorb cadmium and lead ions from a liquid stream.     

Susceptibility of the Coffee Leaf Miner (Perileucoptera spp.) to Bacillus thuringiensisδ-Endotoxins: A Model for Transgenic Perennial Crops Resistant to Endocarpic Insects

Binding of several Bacillus thuringiensisδ-endotoxins was studied on histological midgut sections of larvae of coffee leaf miner Perileucoptera coffeella from Brazil and Perileucoptera sp from Madagascar. CryIA(a), CryIA(b), CryIA(c), CryIB, CryIE, and CryIIA were tested for binding, and only CryIA(c), CryIB, and CryIE yielded a positive response. The toxins bound to the whole midgut, and the result was identical on both insect populations. The same toxins, to the number of which CryIC was added, were tested on larvae of P. coffeella. CryIA(c) and CryIB were toxic with an LC50 of 1.47 μg/ml and 21.93 μg/ml, respectively. CryIE was not toxic to P. coffeella. CryIA(c) and CryIB were tested for synergistic activity and were shown to act by cumulative effect when delivered to the insect larvae as a mixture. Received: 30 July 1997 / Accepted: 26 August 1997     

Zinc bone loss in chronic renal failure and chronic metabolic acidosis

The effects of chronic metabolic acidosis (CMA) on zinc (Zn) bone content and urinary excretion were examined in the presence of normal or reduced renal function together with some aspects of calcium (Ca) metabolism. Four groups of rats were compared. All were fed a 30% protein and 9 mg Zn/100 g diet. Two were uremic (U): The first developed acidosis (UA), which was suppressed in the other (UNA) by NaHCO₃ supplement. Two other groups had normal renal function: One was normal (CNA), and the other had NH₄Cl in the drinking water and acidosis (CA). Femur total Zn and Ca content was markedly reduced by CMA and was not affected by uremia. Zn urinary excretion was increased by CMA and unaltered by uremia. Ca urinary excretion was markedly reduced in uremic rats, but was enhanced in both acidotic conditions. Urinary Ca and Zn showed a strong correlation in uremic and in control rats. Plasma parathormone and 1,25(OH)₂D₃ were unchanged by CMA. These data are in agreement with a direct primary effect of CMA on bone in releasing buffers. CMA induces bone resorption and a parallel decrease of mineral bone components, such as Ca and Zn, with little or no role of PTH, 1,25(OH)₂D₃ and of uremia itself.     

Long-Term Monitoring of Waterborne Pathogens and Microbial Source Tracking Markers in Paired Agricultural Watersheds under Controlled and Conventional Tile Drainage Management

Surface waters from paired agricultural watersheds under controlled tile drainage (CTD) and uncontrolled tile drainage (UCTD) were monitored over 7 years in order to determine if there was an effect of CTD (imposed during the growing season) on occurrences and loadings of bacterial and viral pathogens, coliphages, and microbial source tracking markers. There were significantly lower occurrences of human, ruminant, and livestock (ruminant plus pig) Bacteroidales markers in the CTD watershed in relation to the UCTD watershed. As for pathogens, there were significantly lower occurrences of Salmonella spp. and Arcobacter spp. in the CTD watershed. There were no instances where there were significantly higher quantitative loadings of any microbial target in the CTD watershed, except for F-specific DNA (F-DNA) and F-RNA coliphages, perhaps as a result of fecal inputs from a hobby farm independent of the drainage practice treatments. There was lower loading of the ruminant marker in the CTD watershed in relation to the UCTD system, and results were significant at the level P = 0.06. The odds of Salmonella spp. occurring increased when a ruminant marker was present relative to when the ruminant marker was absent, yet for Arcobacter spp., the odds of this pathogen occurring significantly decreased when a ruminant marker was present relative to when the ruminant marker was absent (but increased when a wildlife marker was present relative to when the wildlife marker was absent). Interestingly, the odds of norovirus GII (associated with human and swine) occurring in water increased significantly when a ruminant marker was present relative to when a ruminant marker was absent. Overall, this study suggests that fecal pollution from tile-drained fields to stream could be reduced by CTD utilization.     

Electroporation of embryogenic protoplasts of sweet orange (<Emphasis Type="Italic">Citrus sinensis</Emphasis> (L.) Osbeck) and regeneration of transformed plants

Summary Electroporation conditions were optimized for the transfection of protoplasts isolated from an embryogenic cell line of sweet organe [Citrus sinensis (L.) Osbeck ev. Hamlin]. Electric field strength (375–450 V cm⁻¹) vector DNA concentration (100 μgml⁻¹), carrier DNA concentration (100 μgml⁻¹), electroporation buffer (pH 8), and preelectroporation heat shock of protoplasts (5 min at 45°C) were optimized. The plasmid vector pBI221 containing the β-glucuronidase (GUS) coding sequence under the control of the CaMV 35S promoter was used and GUS activity was measured 24h after electroporation. All variables significantly affected transfection efficiency and when optimal conditions for each were combined. GUS activity was 7714 pmol 4-methylumbelliferone (MU) mg⁻¹ (protein) min⁻¹. Protoplasts were then electroporated in the presence of green fluorescent protein (GFP) expression vectors pARS101 or pARS108. Green fluorescent embryos were selected, plants regenerated, and integration of the transgene was confirmed by Southern blot analysis. Both plasmids were constructed using EGFP, a GFP variant 35 times brighter than wtGFP, having a single, red-shifted excitation peak, and optimized for human codon-usage. pARS101 was constructed by placing EGFP under the control of a 35S–35S promoter containing 33 bp of the untranslated leader sequence from alfalfa mosaic virus. pARS108 was constructed similarly except sequences were added for transport and retention of EGFP in the lumen of the endoplasmic reticulum. Mention of a trademark, warranty, proprietary product, or vendor does not constitute a guarantee by the US Department of Agriculture and does not imply its approval to the exclusion of other products or veudors that may also be suitable.     

Calf-Level Factors Associated with Bovine Neonatal Pancytopenia – A Multi-Country Case-Control Study

Bovine neonatal pancytopenia (BNP), a high fatality condition causing haemorrhages in calves aged less than 4 weeks, was first reported in 2007 in Germany and subsequently observed at low incidence in other European countries and New Zealand. A multi-country matched case-control study was conducted in 2011 to identify calf-level risk factors for BNP. 405 BNP cases were recruited from 330 farms in Belgium, France, Germany and the Netherlands by laboratory confirmation of farmer-reported cases. Up to four calves of similar age from the same farm were selected as controls (1154 calves). Risk factor data were collected by questionnaire. Multivariable modelling using conditional logistic regression indicated that PregSure®BVD (PregSure, Pfizer Animal Health) vaccination of the dam was strongly associated with BNP cases (adjusted matched Odds Ratio - amOR 17.8 first lactation dams; 95% confidence interval – ci 2.4, 134.4; p = 0.005), and second or more lactation PregSure-vaccinated dams were more likely to have a case than first lactation vaccinated dams (amOR 2.2 second lactation; ci 1.1, 4.3; p = 0.024; amOR 5.3 third or more lactation; ci 2.9, 9.8; p = <0.001). Feeding colostrum from other cows was strongly associated with BNP if the dam was not PregSure-vaccinated (amOR 30.5; ci 2.1, 440.5; p = 0.012), but the effect was less if the dam was PregSure-vaccinated (amOR 2.1; ci 1.1, 4.0; p = 0.024). Feeding exclusively dam’s milk was a higher risk than other types of milk (amOR 3.4; ci 1.6, 7.5; p = 0.002). The population attributable fractions were 0.84 (ci 0.68, 0.92) for PregSure vaccination, 0.13 (ci 0.06, 0.19) for feeding other cows’ colostrum, and 0.15 (ci 0.08, 0.22) for feeding dam’s milk. No other calf-level factors were identified, suggesting that there are other important factors that are outside the scope of this study, such as genetics, which explain why BNP develops in some PregSure-colostrum-exposed calves but not in others.