Spironucleus meleagridis, an enteric diplomonad protozoan of cockatiels (Nymphicus hollandicus): preliminary molecular characterization and association with clinical disease.

A flagellated enteric diplomonad protozoan consistent with Spironucleus meleagridis (formerly Hexamita meleagridis) associated with gastrointestinal disease and mortality in psittacine birds including cockatiels (Nymphicus hollandicus) has been sporadically described in the literature. However, molecular characterization of psittacine protozoal isolates had not yet been performed. The 16S rRNA gene from a protozoan persistently shed in the feces in a small group of cockatiels demonstrated a 98% molecular identity with S. meleagridis isolated from turkeys. Based on these sequence data, a diagnostic PCR assay was developed to detect the presence of S. meleagridis. Nineteen privately owned pet cockatiels from unrelated households were clinically evaluated. All birds microscopically positive for this organism were PCR positive, with several additional birds microscopically negative but PCR positive. Many of the birds identified as positive for S. meleagridis by fecal PCR had signs of gastrointestinal disease such as diarrhea, soft feces, and melena, whereas none of the birds that tested negative had gastrointestinal signs. Examination of feces from two unrelated cockatiel breeding facilities revealed 70% and 86% PCR positive rates. Prevalence of infection and incidence of clinical disease, including factors that lead to clinical manifestation such as viral, bacterial, or mycotic coinfections, are not yet known and warrant further study, but spironucleosis is likely an under-recognized disease in cockatiels.

Investigation of a commercial ELISA for the detection of canine procalcitonin.

BACKGROUND: Rapid identification of sepsis enables prompt administration of antibiotics and is essential to improve patient survival. Procalcitonin (PCT) is a biomarker used to diagnose sepsis in people. Commercial assays to measure canine PCT peptide have not been validated. OBJECTIVE: To investigate the validity of a commercially available enzyme-linked immunosorbent assay (ELISA) marketed for the measurement of canine PCT. ANIMALS: Three dogs with sepsis, 1 healthy dog, 1 dog with thyroid carcinoma. METHODS: Experimental study. The ELISA's ability to detect recombinant and native canine PCT was investigated and intra-assay and interassay coefficients of variability were calculated. Assay validation including mass spectrometry of the kit standard solution was performed. RESULTS: The ELISA did not consistently detect recombinant canine PCT. Thyroid lysate yielded a positive ELISA signal. Intra-assay variability ranged from 18.9 to 77.4%, while interassay variability ranged from 56.1 to 79.5%. Mass spectrometry of the standard solution provided with the evaluated ELISA kit did not indicate presence of PCT. CONCLUSIONS AND CLINICAL IMPORTANCE: The results of this investigation do not support the use of this ELISA for the detection of PCT in dogs.

Diminazene diaceturate for treatment of chronic Cytauxzoon felis parasitemia in naturally infected cats.

BACKGROUND: Cytauxzoon felis is a hemoprotozoal parasite that causes substantial morbidity and mortality during the acute phase of infection in cats. However, cats that survive the acute illness remain persistently infected and may serve as a reservoir for the tick-transmitted pathogen. OBJECTIVE: We investigated the ability of the antiprotozoal compound diminazene diaceturate to eliminate the pathogen from naturally infected C. felis carriers. ANIMALS: Seven healthy, chronically infected domestic cats housed in a research setting. METHODS: Prospective clinical trial. Cats were treated in a masked fashion with diminazene diaceturate (3 mg/kg) or placebo IM in a series of 2 injections 7 days apart. Clearance of the organism was assessed by light microscopy and real-time polymerase chain reaction (PCR) at 0, 3, 6, and 10 weeks. In addition, cats were monitored for behavioral changes or for changes on physical examination, CBC, plasma biochemical profile, and urinalysis periodically. Cats that remained parasitemic at the end of 10 weeks were switched to the alternative treatment and similarly monitored for an additional 10 weeks. RESULTS: Adverse events associated with treatment were limited to self-resolving hypersalivation and injection site soreness; the former was ameliorated by premedication with atropine. Parasite burden, as assayed by both light microscopy and real-time PCR, was similar between diminazene- and placebo-treated cats. CONCLUSIONS AND CLINICAL RELEVANCE: Diminazene diaceturate was unable to eliminate the pathogen or decrease parasite burden in healthy, chronically infected cats.

Hypocalcemia and hypovitaminosis D in dogs with induced endotoxemia.

BACKGROUND: Hypocalcemia is a documented electrolyte disturbance in people and animals with sepsis, but its mechanism is poorly understood. OBJECTIVE: To investigate mechanisms of hypocalcemia in dogs with experimentally induced endotoxemia. ANIMALS: Six healthy mixed breed dogs were included in this nonrandomized, placebo-controlled, crossover study. METHODS: Dogs initially were injected with placebo (0.9% NaCl; 1 mL, IV) and then lipopolysaccharide (LPS; 2 µg/kg, IV) after a 5-day washout period. Blood and urine samples were collected for measurement of serum total calcium (tCa), ionized calcium (iCa), total magnesium (tMg), ionized magnesium (iMg), parathyroid hormone (PTH), 25-hydroxyvitamin D (vitamin D), venous blood gases, and fractional excretion (FE) of calcium. RESULTS: After LPS administration, body temperature increased and blood pressure decreased. Both iCa and tCa decreased (P < .01), but iMg was not significantly different between control and LPS treatments. PTH concentrations increased (P < .01) and vitamin D concentrations decreased (P < .01). Venous pH, bicarbonate, base excess, and blood glucose also decreased (P < .01). Urine tCa concentration was below the limit of detection for all dogs after LPS administration. CONCLUSIONS: Hypocalcemia occurs during endotoxemia in dogs and is associated with hypovitaminosis D. Hypomagnesemia, hypoparathyroidism, alkalosis, and increased calciuresis are not associated with hypocalcemia in endotoxemic dogs.

Feasibility of administering zoledronic acid in palliative patients being cared for in the community: results of a pilot study.

Tumour-induced hypercalcemia (TIH) and pain from bone metastases are common complications of advanced malignancy and have a significant negative impact on quality of life. Many cancer patients in the advanced stages of their palliative illness prefer to avoid hospitalization and to receive their care in the community setting. This small open-label prospective pilot study explored the feasibility of administering zoledronic acid intravenously in the community setting (home and residential hospices). It enrolled a convenience sample of 12 patients with advanced cancer and TIH (n = 7), malignant bone pain (n = 3), or TIH and malignant bone pain (n = 2). The mean duration of infusion was 15 minutes (range: 14-30 minutes). The total nursing time required was 95 minutes, and the mean total cost, including nursing time, travel time, and drug costs was $708.97 per infusion. This cost was compared with costs for clodronate and pamidronate ($402.52 and $406.12 respectively). Calcium fell from a mean of 2.97 mmol/L on day 0 to 2.63 mmol/L on day 4 and to 2.54 mmol/L on day 10. Delirium resolved in 2 of 5 patients with TIH-associated delirium. Intravenous zoledronic acid administered in the community to palliative patients at the end of life is feasible and safe, and the short duration of infusion offers advantages to patients and nursing resources alike. The higher cost of zoledronic acid per infusion may be offset by the advantage of its short infusion time.

Bartonella DNA in the blood and lymph nodes of Golden Retrievers with lymphoma and in healthy controls.

BACKGROUND: Although lymphoma is the most common neoplastic process reported in dogs, its precise etiology is unknown. Golden Retrievers are more likely to develop lymphoma, suggesting a breed predisposition; however, other factors, including environment, immunity, and infection, are likely contributors to oncogenesis. HYPOTHESIS: We hypothesized that the development of lymphoma in Golden Retrievers may be associated with vector-borne infections, specifically Bartonella, Anaplasma, or Ehrlichia species infections. ANIMALS: Golden Retrievers with lymphoma and healthy Golden Retrievers from across the United States were recruited for study participation. METHODS: A matched, case-control study was performed to determine the association of lymphoma and the presence of Bartonella, Anaplasma, and Ehrlichia species in serum, blood, and lymph node aspirates. RESULTS: Using PCR analyses and DNA sequencing, single and coinfections with Bartonella henselae, Bartonella elizabethae, Bartonella quintana, and/or Bartonella vinsonii (berkhoffii) were detected in the blood and lymph node aspirates of Golden Retrievers with lymphoma (5/28 dogs, 18%) and in healthy Golden Retrievers (10/56 dogs, 18%); no Anaplasma or Ehrlichia DNA was detected in any dog. When compared with dogs with lymphoma, a higher (P <.001) proportion of healthy Golden Retrievers were receiving monthly acaricide treatments (2.6 times higher). CONCLUSIONS AND CLINICAL IMPORTANCE: Bartonella DNA can be detected in blood and lymph nodes; importantly, in this report, Bartonella was detected in the same proportion of clinically healthy dogs and dogs with lymphoma. Longitudinal studies should be conducted to determine the mode of transmission of Bartonella in dogs, whether lymphatic infection is persistent, or whether these bacteria may contribute to the development of lymphoma.

Molecular evidence of prevalent dual piroplasma infections in North American raccoons (Procyon lotor).

Based on 18S rRNA sequence analyses 2 distinct genotypes of piroplasms have been described in raccoons. One genotype resides in the Babesia sensu stricto clade and the other in the Babesia microti-like clade. Since these organisms appear morphologically indistinguishable, it is unclear which strain is responsible for the majority of the infections in raccoons. In order to overcome these limitations we performed a molecular survey of raccoons using polymerase chain reaction assays specific for each genotype. We tested blood samples from 41 wild raccoons trapped in eastern North Carolina using PCR assays and found that 95% (39/41) had detectable piroplasm DNA. Ninety percent (37/41) of the samples contained Babesia sensu stricto DNA and 83% (34/41) samples contained Babesia microti-like DNA. DNA from both genotypes was present in 76% (31/41) samples suggesting a very high rate of co-infections. The presence of dual piroplasma infections in carnivores appears to be an uncommon finding. This study highlights the need for molecular assays for the accurate identification of piroplasma. Further studies are indicated to investigate the ability of these parasites to infect domestic animals as well as their zoonotic potential.

The identification of a genetically unique piroplasma in North American river otters (Lontra canadensis).

During a routine health check of a wild-caught North American river otter (Lontra canadensis) small piroplasms were noted within erythrocytes. Analyses of the 18S ribosomal ribonucleic acid (rRNA) gene sequences determined that this was a genetically unique organism most closely related to Babesia microti-like parasites found in other small carnivores. Subsequently 39 wild-trapped North American river otters from North Carolina were tested for the presence of piroplasma deoxyribonucleic acid (DNA) via polymerase chain reaction and piroplasma DNA was detected in 82% (32/39) of these samples. Sequencing of partial 18S rRNA genes from selected cases determined that they were identical to the sentinel case. This report documents the existence of a genetically unique piroplasma in North American river otters and indicates that the prevalence of piroplasma in North Carolina otters is quite high. The pathogenic potential of this organism for otters or other species remains unknown.

Factors influencing theoretical knowledge and practical skill acquisition in student nurses: an empirical experiment.

A previous qualitative study [Nurse Education Today 20 (2000) 499] investigated perceptions of nurse teachers, student nurses and preceptors of the theory-practice gap said to exist within nursing. One theme was views of how the theory-practice gap could be closed. A subsequent quantitative study is reported here, in which this theme was translated into three factors. A full factorial experimental design was used to study the effect of these factors on theoretical knowledge and practical skill acquisition in a sample of first year undergraduate student nurses from one institution of higher education (n=19). The effect of whether a nurse teacher or preceptor taught students theoretical elements relating to a clinical specialty, whether the nurse teacher and preceptor collaborated on the content of what was taught to students and whether students went straight to, or delayed the clinical specialty following theoretical input, was examined. The results demonstrated preceptors were more effective than nurse teachers in promoting theoretical knowledge relating to their clinical specialty. Collaboration between the preceptors and nurse teachers on teaching content was ineffective at increasing theoretical knowledge. Delay between theoretical input and clinical experience was not detrimental for medical placements and for rehabilitation placements, resulted in an improved theoretical knowledge.

Testican in human blood.

Testican is a highly conserved, differentially expressed gene product of unknown function. Since testican is expressed by human endothelial cells and includes a signal sequence, it was our hypothesis that testican protein would be present in blood. We have developed chicken antibodies specific for testican sequence near the N-terminal and identified a 130-kDa form of testican in human plasma. This is much larger than the calculated molecular weight of the encoded polypeptide, suggesting glycosylation of this plasma protein, and large forms of recombinant testican produced in culture were found to include chondroitin sulfate. The 130-kDa form of testican is unstable in plasma. It is converted to smaller stable forms by separable plasma factors that can be blocked by certain serine protease inhibitors. Testican size conversion may be important in its functional activation or decay. One testican domain has strong homology to thyropin-type cysteine protease-inhibitors. Thus, testican may have a function related to protease inhibition in the blood.

Food protein-induced enterocolitis syndrome: report of one case.

We report a 76-day old infant who got diarrhea within the first week of life. He was treated as acute gastroenterocolitis and kept on feeding with regular infant formula. Because the symptoms persisted, the feeding formula was shifted to soy-based formula then to the highly-hydrolyzed formula and got improvement. But severe bloody diarrhea, vomiting, dehydration and fever developed after feeding with regular infant formula again. Based on the history and clinical presentations, cow's milk allergy was suspected. He received total parenteral nutrition for 5 days then fed with highly-hydrolyzed formula with slowly increasing amount. Thereafter tests for total eosinophil counts, total serum IgE, milk specific IgE antibodies and milk extract skin prick test were all unremarkable. Under the impression of food protein-induced enterocolitis syndrome (FPIES), a double-blind placebo-controlled food challenge (DBPCFC) with infant formula was performed. Regular infant formula induced severe vomiting, diarrhea, fever, acidosis and elevation of absolute neutrophil counts (ANC) of peripheral blood by 27,640/mm3. Based on the laboratory findings and challenge results, the patient fit the diagnostic criteria of food protein-induced enterocolitis syndrome.

Distribution of testican expression in human brain.

Testican is a putative extracellular heparan/ chondroitin sulfate proteoglycan of unknown function that is expressed in a variety of human tissues at widely different levels but is most abundant in the brain. In mice, testican mRNA has been detected only in brain and it is therefore likely to have an important function in the central nervous system. RNA blot analysis reveals the relative intensity of testican in various regions of the human brain. Levels of testican message are most pronounced in the thalamus, hippocampus, occipital lobe, nucleus accumbens, temporal lobe, and caudate nucleus, with somewhat lower levels in the cerebral cortex, medulla oblongata, frontal lobe, amygdala, putamen, spinal cord, substantia nigra, and cerebellum. In situ hybridization reveals the cellular distribution of the mRNA within these areas to be highest in neurons and in choroid plexus epithelium, and moderately lower in ependymal cells lining the ventricles and in vascular endothelial cells. Testican mRNA is not detected in oligodendrocytes or in most astrocytes. However, astrocytes in regions of reactive gliosis do express testican mRNA. These findings, along with a cysteine-rich pattern similarity to neurocan, brevican, versican, and other proteoglycans found in brain, suggest that testican may be a part of the specialized extracellular matrix of the brain.

Efficient gene transfer to human endothelial cells using DNA complexed to adenovirus particles.

Human endothelial cells have been found to be relatively refractory to various methods of DNA transfection currently in common use. By using a transfection method involving DNA complexed with replication-deficient adenovirus particles, we have shown that 20% of a population of cultured endothelial cells can be transfected and high levels of transient expression achieved. Both early-passage human umbilical vein endothelial cells and the continuous differentiated line of human endothelium-derived EA.hy926 cells are responsive to this method of transfection. Efficient DNA transfection of endothelial cells is important for studies of endothelium-specific promoters and is a potentially useful route for transgenic therapy.

Endothelial cell expression of testican mRNA.

By screening random cDNAs from a continuous vascular endothelial cell line, EA.hy926, we identified a 5 kb mRNA that is expressed at high levels by this human cell line and by an early passage umbilical vein endothelial cell line. It is detected at lower levels in certain stromal cell lines, but it is not detected in most other cell lines tested, indicating that it represents a differentially expressed function rather than a ubiquitous or housekeeping function. This mRNA was readily detected in samples derived from most human organs as might be expected for a gene expressed in the vascular wall. Sequencing of the 5 kb mRNA reveals its identity with 3.5 kb of previously published testis-derived cDNA sequence called testican (Alliel et al., 1993). Differential expression of this gene by endothelial cells contributes a new perspective on the potential function of testican.

Further studies of the antiemetic activity of granisetron against whole body X-irradiation or cisplatin-induced emesis in the ferret.

In ferrets, the highly selective 5-HT3 receptor antagonist, granisetron, abolished or reduced emesis induced by cisplatin (10 mg/kg i.v.) or whole body X-irradiation (50 Gy in 10.4 min) in a dose-dependent manner when administered by a variety of routes (intravenous, per os, subcutaneous, intramuscular). Complete protection from vomiting and retching was achieved with 0.5 mg/kg i.v. or p.o. granisetron. Granisetron (0.5 mg/kg i.v.) was also effective when given 6 h before cisplatin, completely protecting 50% of ferrets for a total of 10 h. Following repeat dosing, for either 4 days i.v. or 10 days p.o. before emetic challenge, granisetron (0.5 mg/kg) still retained its antiemetic activity on the 5th or 11th day. Prior treatment with cyclophosphamide (80 mg/kg i.v.) resulted in a significantly shorter time to the onset of vomiting after exposure to X-irradiation. Granisetron, but not saline, abolished vomiting and nausea when given as intervention after this combined emetic regimen. These results show that granisetron has potential flexibility for administration via a variety of different routes and also a long duration of action when used as an antiemetic against a wide range of cytostatic agents.

The antiemetic activity of granisetron against cytostatic-treatment-induced emesis in 10- to 13-week-old ferrets.

The antiemetic activity of granisetron was examined in ferrets aged 10-13 weeks. Emesis was induced by exposure to either whole-body X-irradiation (50 Gy over 10.4 min) or cyclophosphamide (80 mg/kg i.v.) plus doxorubicin (6 mg/kg i.v.). Following exposure to whole-body X-irradiation, the young ferrets vomited with a similar latency to vomit and severity of emesis to that shown by adult animals. Granisetron (0.5 mg/kg i.v.) significantly reduced (P < or = 0.05) the number of vomits and retches and two out of four animals were completely protected. Following injection of cyclophosphamide and doxorubicin, the young ferrets showed a reduced emetic response compared to adult animals. Following a dose of granisetron (0.5 mg/kg i.v.), only one out of four ferrets vomited compared to four out of four in the control group. Further experiments showed that cisplatin (12.5 mg/kg i.v.) was unable to induce vomiting in the young ferret (n = 2). Granisetron (0.5 mg/kg i.v.) was well tolerated by the young ferret and was able to reduce significantly or completely abolish emesis induced by cytostatic treatment. The data support the use of granisetron in pediatric patients and clinical trials are currently underway in this patient population.

Extent of differentiated gene expression in the human endothelium-derived EA.hy926 cell line.

Vascular endothelial cells perform many differentiated functions in processes such as angiogenesis, hemostasis, and inflammation. The number of recognized differentiated functions has increased rapidly in recent years, but there may be many more still unrecognized. The purpose of this study is to estimate the fraction of differentially expressed mRNA in a continuous human endothelium-derived cell line, EA.hy926. Random cDNA clones representing mRNAs from this cell line were labeled and used to probe blots of RNA from EA.hy926 cells and from cells of a relatively undifferentiated line. Of 49 random cDNAs, 5 cDNAs or 10% were found to represent mRNAs that are differentially expressed in EA.hy926 and in early passage umbilical vein endothelial cells. Since more than 10(4) different genes are thought to be expressed in the typical mammalian cell, our data indicate that about 10(3) gene products contribute to the differentiated properties of endothelial cells.

Are all 5-HT3 receptor antagonists the same?

A number of 5-HT3 receptor antagonists are currently in clinical development as antiemetics. In this paper we focus on two of these antagonists, granisetron and ondansetron, and compare their antimetic activity against cisplatin (10 mg/kg i.v.)- or whole body X-irradiation (200 rads)-induced emesis in the conscious ferret. The results presented here have been discussed in the light of the recently published literature. Our data suggest that in comparison to ondansetron, granisetron is a more potent, longer acting and pharmacologically "cleaner" compound with a more conventional dose-response profile. The possible impact of these features upon the performance of these compounds in the clinic is discussed particularly with respect to dosing regimens and clinical efficacy. Differences appear to be emerging between granisetron and ondansetron in both these respects, although a direct head-to-head clinical comparison has yet to be carried out. This would involve studies monitoring a sufficiently high number of patients receiving severely emetogenic regimes to allow real clinical differences to be detected with the appropriate statistical power.