Pharmacokinetics of intravenous lithium chloride and assessment of agreement between two methods of lithium concentration measurement in the horse.

BACKGROUND: Pharmacokinetics of lithium chloride (LiCl) administered as a bolus, once i.v. have not been determined in horses. There is no point-of-care test to measure lithium (Li+ ) concentrations in horses in order to monitor therapeutic levels and avoid toxicity. OBJECTIVES: To determine the pharmacokinetics of LiCl in healthy adult horses and to compare agreement between two methods of plasma Li+ concentration measurement: spectrophotometric enzymatic assay (SEA) and inductively coupled plasma mass spectrometry (ICP-MS). STUDY DESIGN: Nonrandomised, single exposure with repeated measures over time. METHODS: Lithium chloride was administered (0.15 mmol/kg bwt) as an i.v. bolus to eight healthy adult horses. Blood samples were collected pre-administration and at multiple times until 48 h post-administration. Samples were analysed by two methods (SEA and ICP-MS) to determine plasma Li+ concentrations. Pharmacokinetics were determined based on the reference ICP-MS data. RESULTS: Adverse side effects were not observed. The SEA showed linearity, R2 = 0.9752; intraday coefficient of variation, 2.5%; and recovery, 96.3%. Both noncompartmental and compartmental analyses (traditional two-stage and nonlinear mixed-effects [NLME] modelling) were performed. Geometric mean values of noncompartmental parameters were plasma Li+ concentration at time zero, 2.19 mmol/L; terminal elimination half-life, 25.68 h; area under the plasma concentration-time curve from time zero to the limit of quantification, 550 mmol/L min; clearance, 0.273 mL/min/kg; mean residence time, 31.22 h; and volume of distribution at steady state, 511 mL/kg. Results of the traditional two-stage analysis showed good agreement with the NLME modelling approach. Bland-Altman analyses demonstrated poor agreement between the SEA and ICP-MS methods (95% limits of agreement = 0.14 ± 0.13 mmol/L). MAIN LIMITATIONS: Clinical effects of LiCl have not been investigated. CONCLUSIONS: The LiCl i.v. bolus displayed pharmacokinetics similar to those reported in other species. The SEA displayed acceptable precision but did not agree well with the reference method (ICP-MS). The Summary is available in Spanish - see Supporting Information.

A murine model of early Pseudomonas aeruginosa lung disease with transition to chronic infection.

Pseudomonas aeruginosa (PA) remains an important pathogen in patients with cystic fibrosis (CF) lung disease as well as non-CF bronchiectasis and chronic obstructive airways disease. Initial infections are cleared but chronic infection with mucoid strains ensues in the majority of CF patients and specific interventions to prevent this critical infection transition are lacking. The PA bead model has been widely used to study pulmonary P.aeruginosa infection but has limitations in animal husbandry and in accurately mimicking human disease. We have developed an adapted agar bead murine model using a clinical mucoid strain that demonstrates the key features of transition from transitory to chronic airways infection. Infected animals show very limited acute morbidity and mortality, but undergo infection-related weight loss and neutrophilic inflammation, development of anti-pseudomonal antibodies, variable bacterial clearance, endobronchial infection and microbial adaptation with PA small colony variants. We anticipate this model will allow research into the host and microbial factors governing this critical period in Pseudomonas aeruginosa pulmonary pathogenesis when transition to chronicity is occurring.

Lymphocytic foci in the endometrium of pregnant dairy cows: Characterization and association with reduced placental weight and embryonic loss.

Lymphocytic foci (also known as lymphoid aggregates or tertiary lymphoid structures) form within the bovine endometrium after antigenic challenge. Their presence in the pregnant uterus provides evidence for a chronic inflammatory condition perhaps arising from an early postpartum uterine infection. The chronic inflammation that includes the foci could explain greater embryonic loss in dairy cows with early postpartum uterine disease. The objectives were to characterize the size and location of the foci in the pregnant uterus, determine their composition using immunohistochemistry, and associate their presence with the development of the pregnancy and embryonic loss. Pregnant cows (n = 43) were slaughtered on days 28, 35, or 42 of pregnancy. Uterine tissue was collected and processed for histologic and immunohistochemical analysis. The number of small (<100 micron diameter), intermediate (100-250 micron diameter), and large (>250 micron diameter) foci was counted. The number of cows averaging 0, 0.1 to 1, 1.1 to 2, and more than 2 foci per section (small, intermediate, and large; combined) was 7 (16%), 14 (33%), 11 (26%), and 11 (26%), respectively. The average number of small and intermediate foci found in the histologic sections was greater in cows with evidence of uterine infection postpartum (P < 0.05). Lymphocytic foci were distributed within the caruncle and the intercaruncular tissue and comprised a core of CD3-positive cells (T cells) surrounding CD79-positive cells (B cells). The number of lymphocytic foci was correlated with a total inflammation score (on the basis of the total number of inflammatory cells in the endometrium; r(2) = 0.49; P < 0.001) and a fibrosis score (based on the extent of fibrosis in the endometrium; r(2) = 0.33; P < 0.001). Cows with a high foci count (averaging more than 0.5 foci per section) had lesser (P < 0.01) placental weight on Day 42 of pregnancy. There was no effect of foci count on placental weight on Day 28 or 35. Two cows with embryonic loss were in the highest quartile for foci count. In conclusion, cows with chronic inflammation as evidenced by a large number of lymphocytic foci had reduced placental weight during pregnancy. The number of foci in pregnant cows was associated with early postpartum uterine disease. Whether the foci themselves are inhibitory to pregnancy development or are associated with other bacteriological, morphological, or biochemical changes to the uterus that lead to infertility will need to be investigated.

White snakeroot poisoning in goats: Variations in toxicity with different plant chemotypes.

Tremetone and possibly other benzofuran ketones are believed to be the toxic compounds in white snakeroot. However, disease has not been reproduced with purified toxins and the concentrations of the benzofuran ketones in white snakeroot populations that cause toxicosis have not been documented. The objectives of this study were to compare the toxicity of seven plant populations, better characterize the clinical and pathologic changes of poisoning, and correlate intoxication with benzofuran ketone content. Four of the seven white snakeroot collections were toxic at the dose and duration used in the study. Affected goats became exercise intolerant, had significant serum enzyme changes and histological lesions in the large appendicular muscles. The incidence and severity of poisoning was not correlated with total doses of tremetone or total benzofuran ketone concentrations suggesting they may not be closely involved in producing toxicity and the possible involvement of an unidentified toxin. The results also demonstrate that white snakeroot populations vary chemically and toxicologically.

Copper toxicosis in New Zealand White rabbits (Oryctolagus cuniculus).

Six 12- to 14-month-old New Zealand White rabbits were diagnosed with copper toxicosis. These rabbits were part of a group of 110 purchased and shipped overnight for research purposes. On arrival, the group experienced an abrupt diet change. Eight died over 3 weeks and 6 were submitted for postmortem examination. Microscopic findings included severe centrilobular to midzonal hepatocellular necrosis with rhodanine stain-positive copper granules in the remaining hepatocytes. Mild periportal fibrosis and biliary hyperplasia, hemoglobinuric nephrosis, and splenic erythrophagocytosis were also observed. Hepatic copper concentrations were elevated, ranging from 319 to 997 ppm. Clinical disease was not previously observed in younger rabbits gradually transitioned from the supplier's copper-supplemented diet. Copper toxicosis likely occurred in these rabbits from a combination of (1) increased duration of copper supplementation leading to increased hepatocellular stores and (2) stress leading to anorexia and release of hepatocellular copper stores similar to chronic copper toxicosis as described in sheep.

Management of suspected Lyme borreliosis: experience from an outpatient parenteral antibiotic therapy service.

OBJECTIVES: Lyme borreliosis (LB) is the most common human tick-borne infection in Europe and the USA. In this study we set out to analyse the outcome of patients treated for Lyme disease via outpatient parenteral antibiotic therapy (OPAT) and the appropriateness of this treatment using current guidelines. METHODS: This was a retrospective review of all patients with suspected LB managed via OPAT in Glasgow in 2000-11. RESULTS: Of 72 patients treated for suspected LB, 35 patients (49%) were treated in accordance with guidelines and 36 (50%) were treated with no specific guidelines. A definite improvement was seen in 20 patients (28%). Adverse reactions were documented in 29 (40%) patients with neutropenia, and mild liver function derangement was most commonly observed. CONCLUSION: These results show the complexity of translating well-substantiated regimens from clinical trials to actual clinical practice. OPAT was an effective way of administering parenteral therapy for Lyme disease but should not be undertaken lightly due to the rate of adverse events and low rates of success in certain patient groups seen in this study. In view of this, stricter criteria for inclusion to OPAT in line with published guidance should be applied to minimize patient harm and optimize success.

Use of different levels of ground endophyte-infected tall fescue seed during heat stress to separate characteristics of fescue toxicosis.

Studies of fescue toxicosis using whole seed diets show reduced feed intake and thermoregulatory ability, but much of the seed passes undigested through the animal. Cattle were fed ground tall fescue seed at different levels to potentially facilitate digestion and absorption of toxins and identify toxin sensitivity for major characteristics of the condition [i.e., hyperthermia, reduced feed intake (FI), reduced blood prolactin]. Steers (n = 18; 350 kg BW) were housed in the Brody Climatology Laboratory at thermoneutrality (TN; 19°C) and randomly assigned to daily diet treatments with either ground endophyte-infected [E+; low and high doses at 20 and 40 µg ergovaline/(kg BW/d), respectively] or endophyte-free [E-; control at 0 µg ergovaline/(kg BW/d)] tall fescue seed. After 12 d at TN, animals received 2 d of transition to heat stress (HS; 36°C daytime, 25°C nighttime) and maintained for 14 more days. Cattle were fed twice daily at 0800 and 1600 h, with water ad libitum. Feed intake was measured at 0700 h, with skin and rectal temperatures, and respiration rate at 0600, 1100, 1600, and 2100 h. Blood was sampled on selected days for prolactin and leptin determinations. Steers fed ground E+ diet decreased (P ≤ 0.0001) FI below controls at TN, with no dose effect. Maximum FI reduction with E+ treatment was 25% at TN, with an additional 46% decrease during HS (P ≤ 0.05). By the end of HS, E+ FI increased (P > 0.05) to that of E-, suggesting recovery. Prolactin was reduced (P ≤ 0.05) in high E+ cattle below controls at study end. Leptin blood concentrations were unaffected by E+ treatment (P > 0.05) but was reduced (P ≤ 0.05) by the end of HS. Pattern of rectal temperature response to HS showed a more rapid initial increase and decline for both E+ groups compared with controls (P ≤ 0.05). Skin temperature was the only variable that identified E+ dose differences. Although there were no treatment differences at TN, skin temperature was lower (P ≤ 0.05) for high E+ steers compared with controls during HS when air temperature was reduced each day. In general, FI was more responsive to E+ toxins than body temperature or blood prolactin, declining even at TN and exhibiting dynamic activity during HS. Although body temperature response to E+ toxins appears to stabilize during HS, this is misleading as rapid change in air temperature exposes effects on skin temperature.

Phage-selected lipopolysaccharide mutants of Pectobacterium atrosepticum exhibit different impacts on virulence.

AIMS: To positively select Pectobacterium atrosepticum (Pa) mutants with cell surface defects and to assess the impact of these mutations on phytopathogenesis. METHODS AND RESULTS: Several phages were isolated from treated sewage effluent and were found to require bacterial lipopolysaccharide (LPS) for infection. Two strains with distinct mutations in LPS were obtained by transposon mutagenesis. Along with a third LPS mutant, these strains were characterized with respect to various virulence-associated phenotypes, including growth rate, motility and exoenzyme production, demonstrating that LPS mutations are pleiotropic. Two of the strains were deficient in the synthesis of the O-antigen portion of LPS, and both were less virulent than the wild type. A waaJ mutant, which has severe defects in LPS biosynthesis, was dramatically impaired in potato tuber rot assays. The infectivity of these novel phages on 32 additional strains of Pa was tested, showing that most Pa isolates were sensitive to the LPS-dependent phages. CONCLUSIONS: Native LPS is crucial for optimal growth, survival and virulence of Pa in vivo, but simultaneously renders such strains susceptible to phage infection. SIGNIFICANCE AND IMPACT OF THE STUDY: This work demonstrates the power of phages to select and identify the virulence determinants on the bacterial surface, and as potential biocontrol agents for Pa infections.

Exploitation of a new flagellatropic phage of Erwinia for positive selection of bacterial mutants attenuated in plant virulence: towards phage therapy.

AIMS: To isolate and characterize novel bacteriophages for the phytopathogen, Erwinia carotovora ssp. atroseptica (Eca), and to isolate phage-resistant mutants attenuated in virulence. METHODS AND RESULTS: A novel flagellatropic phage was isolated on the potato-rotting bacterial species, Eca, and characterized using electron microscopy and restriction analysis. The phage, named PhiAT1, has an icosahedral head and a long, contractile tail; it belongs to the Myoviridae family. Partial sequencing revealed the presence of genes with homology to those of coliphages T4, T7 and Mu. Phage-resistant transposon mutants of Eca were isolated and studied in vitro for a number of virulence-related phenotypes; only motility was found to be affected. In vivo tuber rotting assays showed that these mutants were attenuated in virulence, presumably because the infection is unable to spread from the initial site of inoculation. CONCLUSIONS: The Eca flagellum can act as a receptor for PhiAT1 infection, and resistant mutants are enriched for motility and virulence defects. SIGNIFICANCE AND IMPACT OF THE STUDY: PhiAT1 is the first reported flagellatropic phage found to infect Eca and has enabled further study of the virulence of this economically important phytopathogen.

Characterization of a broad-host-range flagellum-dependent phage that mediates high-efficiency generalized transduction in, and between, Serratia and Pantoea.

A phage (PhiOT8) isolated on Serratia sp. ATCC 39006 was shown to be flagellum-dependent, and to mediate generalized transduction with high efficiency (up to 10(-4) transductants per p.f.u.). PhiOT8 was shown to have a broad host range because it also infected a strain of Pantoea agglomerans isolated from the rhizosphere. Transduction of plasmid-borne antibiotic resistance between the two bacterial genera was demonstrated, consistent with purported ecological roles of phages in dissemination of genes between bacterial genera. Serratia sp. ATCC 39006 and P. agglomerans produce a number of interesting secondary metabolites that have potential applications in cancer therapy and biocontrol of fungal infections. PhiOT8 has utility as a powerful functional genomics tool in these bacteria.

Effects of short-term heat stress on endophytic ergot alkaloid-induced alterations in rat hepatic gene expression.

Exposure to ergot alkaloids in endophyte-infected fescue (E+) is associated with impaired animal productivity, especially during heat stress, which is commonly referred to as fescue toxicosis. To elucidate the pathogenesis of this condition, the effects of short-term heat stress (HS) on hepatic gene expression in rats exposed to endophytic ergot alkaloids were evaluated. Rats implanted with telemetric transmitters to continuously measure core temperature were fed an E+ diet and maintained under thermoneutral (TN) conditions (21 degrees C) for 5 d, followed by TN or 31 degrees C (HS) conditions for 3 d. Feed intake (FI) and BW were monitored daily. The E+ and HS-induced alterations in hepatic genes were evaluated using DNA microarrays and PCR analyses. Hepatic antioxidant enzyme activities, as well as the incidence of apoptosis, were determined. As expected, intake of E+ reduced FI and BW from pretreatment levels under TN conditions, with greater reductions during short-term HS. Genes involved in gluconeogenesis and apoptosis were upregulated, whereas genes associated with oxidative phosphorylation, xenobiotic metabolism, antioxidative mechanisms, immune function, cellular proliferation, and chaperone activity were all downregulated with short-term HS. Hepatocytic apoptosis was increased and antioxidant enzyme activity decreased in the livers of rats exposed to HS. The hypothesized, exacerbating effects of HS on the direct, endophytic toxin-related and indirect, reduced caloric intake-associated alterations in hepatic gene expression were clearly demonstrated in rats and may help to elucidate the pathogenesis of fescue toxicosis in various animal species.

Equine endometrial fibrosis correlates with 11beta-HSD2, TGF-beta1 and ACE activities.

Endometrial periglandular fibrosis (EPF) contributes to embryonic and fetal loss in mares. Equine EPF correlates inversely with conception and successful gestation. In the modified Kenney endometrial biopsy classification system, EPF categories I, IIA, IIB, and III correspond to minimal, mild, moderate, and severe fibrosis (+/-inflammation), respectively. Paraffin sections of biopsy specimens were stained with H&E, and picrosirius red (specific for fibrillar collagens types I and III), to determine %EPCVF. Endometrial ACE-binding activity, TGF-beta1 and 11beta-HSD2 activities were also measured. Ultrastructural changes in EPF categories IIB and III endometria strongly suggested myofibroblastic transformation. ACE-binding activity was highest in EPF category IIB; however, endometrial TGF-beta1 and 11beta-HSD2 activities were significantly correlated to the severity of EPF (P<0.05). We conclude that, locally generated angiotensin II initiates the expression of TGF-beta1 resulting in myofibroblastic transformation. 11Beta-HSD2 in concert appears to modulate the severity of endometrial fibrosis.

Validation of the in vitro gastrointestinal (IVG) method to estimate relative bioavailable lead in contaminated soils.

The effect of the dosing vehicle (e.g., dough) on the ability of an in vitro gastrointestinal (IVG) method to predict relative bioavailable Pb associated with soil ingestion was evaluated. Bioaccessible Pb determined by the IVG method was compared with relative bioavailable Pb measured from dosing trials using juvenile swine for 18 contaminated soils ranging from 1270 to 14200 mg Pb kg(-1). Bioaccessible Pb was measured in the IVG gastric extraction (GE) and intestinal extraction (IE) solutions. Mean bioaccessible Pb values were 32.2% for GE without dough, 23.0% for GE with dough, 1.06% for IE without dough, and 0.56% for IE with dough. It is possible that phytic acid associated with the dough addition decreased bioaccessible Pb. In vivo relative bioavailable Pb ranges for different swine tissues were 1 to 87% for blood, 0 to 110% for liver, 1 to 124% for kidney, and 0.04 to 94% for bone. Strong linear relationships between IVG GE Pb with dough (r > 0.76, P < 0.0002), IVG IE Pb with dough (r > 0.56, P < 0.015), and IVG GE Pb without dough (r > 0.81, P < 0.0001) and in vivo bioavailable Pb as estimated with blood, kidney, liver, and bone were found. Inexpensive in vitro methods may be useful in providing an estimate of the variability in relative bioavailable Pb at a single study site. The IVG method can be used to estimate relative bioavailable Pb, As, and Cd in contaminated soil.

3-methylindole induces transient olfactory mucosal injury in ponies.

Response to 3-methylindole (3MI) varies among species. Mice recover from 3MI-induced bronchiolar epithelial injury but sustain persistent olfactory mucosal injury with scarring and epithelial metaplasia. In contrast, 3MI induces obliterative bronchiolitis in horses and ponies, but olfactory mucosal injury has not been reported. To evaluate the effect of 3MI on equine olfactory mucosa, ponies were dosed orally with 100 mg 3MI/kg (n = 9) or corn oil vehicle (n = 6). All ponies treated with 3MI developed obliterative bronchiolitis with mild olfactory injury. By 3 days after 3MI dosing, olfactory epithelium appeared disorganized with decreased and uneven surface height and scalloping of the basement membrane zone. Epithelial cells of Bowman's glands were hypertrophic. Proliferation of olfactory epithelium and Bowman's glands was supported by an increased mitotic index and positive immunohistochemical staining for proliferating cell nuclear antigen as compared with controls. The activity of 11beta-hydroxysteroid dehydrogenase, an olfactory mucosal cytosolic enzyme localized to sustentacular and Bowman's glandular epithelial cells, was concurrently decreased. By 9 days postdosing, olfactory mucosal lesions had lessened. Results indicate that 3MI transiently injures equine olfactory mucosa without the extensive necrosis, scarring, or metaplasia seen in murine olfactory mucosa or in equine bronchiolar epithelium.

Macrophage nitric oxide synthase associates with cortical actin but is not recruited to phagosomes.

Nitric oxide (NO) produced from inducible NO synthase (iNOS) is an important component of host defense against intracellular pathogens. To understand how phagocytes deliver NO to ingested microorganisms while avoiding cytotoxicity, we set out to study the subcellular localization of iNOS within macrophages following phagocytosis. Confocal microscopy of immunostained cells showed that iNOS was located not only diffusely within cytoplasm but also in vesicles, as well as immediately adjacent to the peripheral cell membrane. This peripheral iNOS colocalized with the cortical actin cytoskeleton and was removed by the actin-depolymerizing drug cytochalasin B. Biochemical fractionation of RAW 264 macrophages showed that 32.75% (+/-5.11%; n = 3) of iNOS was present in a particulate fraction, which cosedimented with low-density cellular vesicles. Following phagocytosis of latex beads, zymosan, immunoglobulin G-coated beads, or complement-coated zymosan, submembranous cortical iNOS was not recruited to phagosomes, nor was there any relocalization of intracellular iNOS. Similarly, following phagocytosis of Salmonella enterica serovar Typhimurium there was no recruitment of iNOS to the Salmonella vacuole at any stage after internalization. NO mediated significant killing of intracellular S. enterica serovar Typhimurium in RAW macrophages treated with lipopolysaccharide and gamma interferon; this was evident 4 h after infection. Although not recruited to phagosomes, iNOS association with the submembranous cortical actin cytoskeleton is ideally suited to deliver NO to microbes in contact with the cell surface and may contribute to early killing of ingested Salmonella.

Increased expression of inducible nitric oxide synthase and cyclo-oxygenase-2 in the airway epithelium of asthmatic subjects and regulation by corticosteroid treatment.

BACKGROUND: Nitric oxide (NO) and prostanoids are mediators of vascular and bronchial tone that are postulated to be involved in asthma. Increased levels of both are found in asthmatic subjects and are synthesised by enzymes that have cytokine inducible forms: inducible NO synthase (iNOS) and cyclo-oxygenase-2 (COX-2), respectively. We hypothesised that the in vivo expression of iNOS and COX-2 in the airways would be increased in asthma, and that these cytokine inducible enzymes may represent targets for regulation by corticosteroid treatment. METHODS: Bronchial biopsy specimens were obtained from three groups of subjects: atopic asthmatics treated with beta(2) agonists alone (n=7), atopic asthmatics additionally receiving regular treatment with corticosteroids (n=8), and non-asthmatic control subjects (n=10). Expression of iNOS and COX-2 mRNA and immunoreactive protein was studied using in situ hybridisation and quantitative immunohistochemistry. RESULTS: Immunoreactivity and the hybridisation signal for iNOS and COX-2 were mainly localised in the airway epithelium. The proportion of epithelium immunostained was significantly greater in the non-steroid treated asthmatic subjects (iNOS 8.6 (1.8)%; COX-2 26.3 (4.6)%) than either the steroid treated asthmatics (iNOS 3.4 (1.0)%, p=0.009; COX-2 13.0 (0.6)%, p=0.0015) or the non-asthmatic controls (iNOS 4.2 (0.9)%, p=0.018; COX-2 11.6 (0.6)%, p=0.0003). Similarly, the hybridisation signal was stronger in the non-steroid treated group of asthmatic subjects than in the other two groups. CONCLUSIONS: These findings highlight the potential role of the airway epithelium both as a contributor to the inflammatory process in asthma and as a target for inhaled corticosteroid treatment in this disease.