Development of an intrauterine infection model in the postpartum dairy cow.

AIMS: To develop an intrauterine infection model for Trueperella pyogenes in postpartum dairy cows and to assess the effect of this infection on the degree of intrauterine inflammation and concentrations of progesterone in serum. METHODS: The oestrous cycles of 36 healthy, non-pregnant, postpartum dairy cows were synchronised. They were then treated by intrauterine infusion of 0.5 g cephapirin before being blocked by age and randomly assigned to treatment with intrauterine infusion of saline (n = 18), 107 (n = 9) or 109 (n = 9) cfu of T. pyogenes, approximately 4 days after the expected time of ovulation (Day 0). Prior to intrauterine infusion on Day 0 and again on Days 3, 7, 10, and 15, cytobrush samples were collected from the uterus of each cow for microbiology and assessment of the percentage of polymorphonuclear neutrophils (PMN%). Blood samples were collected on the same days for measurement of concentrations of progesterone in serum, and uterine lumen diameter was assessed daily using transrectal ultrasonography. RESULTS: Trueperella pyogenes was isolated from 5/18 (28%), 7/9 (78%) and 8/9 (89%) cows infused with saline, 107 or 109 cfu of T. pyogenes, respectively (p < 0.001). Mean PMN% in the control cows did not change over time (p > 0.05), whereas it was higher on Days 7 and 10 than Day 0 in the 107 cfu group, and higher on Days 3 and 10 than Day 0 in the 109 cfu group (p < 0.05). The percentage of observations with uterine lumen diameters >2 mm was higher in cows infused with 107 (29.3 (95% CI = 14.5-44.2)%) or 109 cfu (19.2 (95% CI = 7.0-31.5)%) than in control cows (3.1 (95% CI = 0.1-6.0)%) (p < 0.001). Mean concentrations of progesterone in serum were higher in cows infused with 107 cfu (2.01 (SE 0.19) ng/mL) than cows infused with 109 cfu (1.01 (SE 0.27) ng/mL), with the control group intermediate (1.41 (SE 0.19) ng/mL) (p = 0.03). CONCLUSIONS: Infusion of 107 or 109 cfu of T. pyogenes resulted in the establishment of intrauterine infection in 83% of cows. Infection resulted in increased uterine lumen diameter, and an inflammatory response, i.e. elevated PMN%. CLINICAL RELEVANCE: This intrauterine infection model may be useful for future research on, for example, the pathogenesis of intrauterine infection in postpartum dairy cows.

The Dynamic Influence of Olorofim (F901318) on the Cell Morphology and Organization of Living Cells of Aspergillus fumigatus.

The first characterized antifungal in the orotomide class is olorofim. It targets the de novo pyrimidine biosynthesis pathway by inhibiting dihydroorotate dehydrogenase (DHODH). The pyrimidines uracil, thymine and cytosine are the building blocks of DNA and RNA; thus, inhibition of their synthesis is likely to have multiple effects, including affecting cell cycle regulation and protein synthesis. Additionally, uridine-5'-triphosphate (UTP) is required for the formation of uridine-diphosphate glucose (UDP-glucose), which is an important precursor for several cell wall components. In this study, the dynamic effects of olorofim treatment on the morphology and organization of Aspergillus fumigatus hyphae were analyzed microscopically using confocal live-cell imaging. Treatment with olorofim led to increased chitin content in the cell wall, increased septation, enlargement of vacuoles and inhibition of mitosis. Furthermore, vesicle-like structures, which could not be stained or visualized with a range of membrane- or vacuole-selective dyes, were found in treated hyphae. A colocalization study of DHODH and MitoTracker Red FM confirmed for the first time that A. fumigatus DHODH is localized in the mitochondria. Overall, olorofim treatment was found to significantly influence the dynamic structure and organization of A. fumigatus hyphae.

Effect of the Novel Antifungal Drug F901318 (Olorofim) on Growth and Viability of Aspergillus fumigatus.

F901318 (olorofim) is a novel antifungal drug that is highly active against Aspergillus species. Belonging to a new class of antifungals called the orotomides, F901318 targets dihydroorotate dehydrogenase (DHODH) in the de novo pyrimidine biosynthesis pathway. In this study, the antifungal effects of F901318 against Aspergillus fumigatus were investigated. Live cell imaging revealed that, at a concentration of 0.1 µg/ml, F901318 completely inhibited germination, but conidia continued to expand by isotropic growth for >120 h. When this low F901318 concentration was applied to germlings or vegetative hyphae, their elongation was completely inhibited within 10 h. Staining with the fluorescent viability dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC) showed that prolonged exposure to F901318 (>24 h) led to vegetative hyphal swelling and a decrease in hyphal viability through cell lysis. The time-dependent killing of F901318 was further confirmed by measuring the fungal biomass and growth rate in liquid culture. The ability of hyphal growth to recover in drug-free medium after 24 h of exposure to F901318 was strongly impaired compared to that of the untreated control. A longer treatment of 48 h further improved the antifungal effect of F901318. Together, the results of this study indicate that F901318 initially has a fungistatic effect on Aspergillus isolates by inhibiting germination and growth, but prolonged exposure is fungicidal through hyphal swelling followed by cell lysis.

Antimicrobial usage and risk of retreatment for mild to moderate clinical mastitis cases on dairy farms following on-farm bacterial culture and selective therapy.

AIMS: To assess antimicrobial usage for treatment of mild to moderate clinical mastitis, and risk of retreatment, following implementation of an on-farm bacterial culture system and selective therapy based on culture results, and to assess compliance with treatment decision tree protocols and the level of agreement between results from on-farm culture and laboratory-based microbiology methods. MATERIALS AND METHODS: Herdowners from seven dairy herds were asked to collect milk samples from cases of mild to moderate clinical mastitis between July 2015 and May 2016. All samples were cultured on-farm using a commercially available selective media and were also submitted for laboratory-based culture. Within sequential pairs of cows with mastitis, half were assigned to be treated without regard to culture results (Blanket group), and half were treated based on the on-farm culture results (Selective group) according to decision tree diagrams provided to the farmers. Culture results, treatments, and retreatments for clinical mastitis were recorded. The sum of the daily doses of antimicrobials used per cow, the number of retreatments and interval to first retreatment were compared between treatment groups. RESULTS: The geometric mean sum of daily doses for quarters assigned to the Selective (1.72 (95% CI=1.55-1.90)) group was lower than for the Blanket (2.38 (95% CI=2.17-2.60)) group (p=0.005). The percentage of cows retreated for clinical mastitis did not differ between the Selective (21.7 (95% CI=10.5-25.9)%) and Blanket (26.1 (95% CI=20.9-31.3)%) groups (p=0.13), and there was no difference between groups in the hazard that cows would be retreated within 60 days of enrolment (hazard ratio=0.82 (95% CI=0.39-1.69); p=0.59). Compliance with the treatment protocols was higher amongst quarters assigned to the Selective (199/233; 85.4%) compared with the Blanket (171/249; 68.7%) group (p<0.001), and varied between farms from 64-94%. The overall agreement between results from on-farm and laboratory culture was 188/331 (56.9%; kappa=0.31; p<0.001), but varied between farms from 44.7-88.2% (p<0.001). CONCLUSIONS: Use of on-farm culture with selective antimicrobial therapy resulted in approximately 25% lower antimicrobial usage, but was not associated with an increase in the proportion of cows retreated for clinical mastitis. CLINICAL RELEVANCE: This study has demonstrated that on-farm culture and selective therapy based on culture results can be implemented on-farm. However, farms varied in their implementation of both the treatment protocols and microbiology procedures. Where such systems are to be used on-farm, specific training and on-going monitoring is required.

F901318 represents a novel class of antifungal drug that inhibits dihydroorotate dehydrogenase.

There is an important medical need for new antifungal agents with novel mechanisms of action to treat the increasing number of patients with life-threatening systemic fungal disease and to overcome the growing problem of resistance to current therapies. F901318, the leading representative of a novel class of drug, the orotomides, is an antifungal drug in clinical development that demonstrates excellent potency against a broad range of dimorphic and filamentous fungi. In vitro susceptibility testing of F901318 against more than 100 strains from the four main pathogenic Aspergillus spp. revealed minimal inhibitory concentrations of ≤0.06 µg/mL-greater potency than the leading antifungal classes. An investigation into the mechanism of action of F901318 found that it acts via inhibition of the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH) in a fungal-specific manner. Homology modeling of Aspergillus fumigatus DHODH has identified a predicted binding mode of the inhibitor and important interacting amino acid residues. In a murine pulmonary model of aspergillosis, F901318 displays in vivo efficacy against a strain of A. fumigatus sensitive to the azole class of antifungals and a strain displaying an azole-resistant phenotype. F901318 is currently in late Phase 1 clinical trials, offering hope that the antifungal armamentarium can be expanded to include a class of agent with a mechanism of action distinct from currently marketed antifungals.

Perinatal inflammation/infection and its association with correction of metabolic acidosis in hypoxic-ischemic encephalopathy.

OBJECTIVE: To investigate the decreased response to hypothermia in neonates with hypoxic-ischemic encephalopathy (HIE) and infection, we sought to determine the association of fetal inflammation/infection with perinatal metabolic acidosis. STUDY DESIGN: We performed a retrospective cohort study of neonates with suspected HIE started on whole-body hypothermia within 6 h of birth that had a cord gas at delivery and placental pathology performed. Neonates were compared based on the presence of clinical and histologic chorioamnionitis. The cord gas at delivery was compared with the initial arterial gas after birth. RESULTS: In all, 50 out of 67 (74.6%) neonates admitted for therapeutic hypothermia met inclusion criteria. Chorioamnionitis did not affect the cord gas at delivery, but both clinical and histologic chorioamnionitis were associated with a significantly increased metabolic acidosis on the initial neonatal arterial gas. CONCLUSION: Chorioamnionitis, diagnosed both clinically and histologically, is associated with a persistent state of acidosis in neonates with HIE that may contribute to worse neurologic outcomes.

Evaluation of a culture-based pathogen identification kit for bacterial causes of bovine mastitis.

Accurate identification of mastitis-causing bacteria supports effective management and can be used to implement selective use of antimicrobials for treatment. The objectives of this study were to compare the results from a culture-based mastitis pathogen detection test kit ('VetoRapid', Vétoquinol) with standard laboratory culture and to evaluate the potential suitability of the test kit to inform a selective treatment programme. Overall 231 quarter milk samples from five UK dairy farms were collected. The sensitivity and specificity of the test kit for the identification of Escherichia coli, Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus uberis and Enterococcus spp. ranged from 17 per cent to 84 per cent and 92 per cent to 98 per cent, respectively. In total, 23 of 68 clinical samples were assigned as meeting the requirement for antimicrobial treatment (Gram-positive organism cultured) according to standard culture results, with the test kit results having sensitivity and specificity of 91 per cent and 78 per cent, respectively. Several occurrences of misidentification are reported, including S. aureus being misidentified as coagulase-negative staphylococci and vice versa. The test kit provides rapid preliminary identification of five common causes of bovine mastitis under UK field conditions and is likely to be suitable for informing selective treatment of clinical mastitis caused by Gram-positive organisms.

The antimicrobial activity of honey against common equine wound bacterial isolates.

Delayed healing associated with distal limb wounds is a particular problem in equine clinical practice. Recent studies in human beings and other species have demonstrated the beneficial wound healing properties of honey, and medical grade honey dressings are available commercially in equine practice. Equine clinicians are reported to source other non-medical grade honeys for the same purpose. This study aimed to assess the antimicrobial activity of a number of honey types against common equine wound bacterial pathogens. Twenty-nine honey products were sourced, including gamma-irradiated and non-irradiated commercial medical grade honeys, supermarket honeys, and honeys from local beekeepers. To exclude contaminated honeys from the project, all honeys were cultured aerobically for evidence of bacterial contamination. Aerobic bacteria or fungi were recovered from 18 products. The antimicrobial activity of the remaining 11 products was assessed against 10 wound bacteria, recovered from the wounds of horses, including methicillin resistant Staphylococcus aureus and Pseudomonas aeruginosa. Eight products were effective against all 10 bacterial isolates at concentrations varying from <2% to 16% (v/v). Overall, the Scottish Heather Honey was the best performing product, and inhibited the growth of all 10 bacterial isolates at concentrations ranging from <2% to 6% (v/v). Although Manuka has been the most studied honey to date, other sources may have valuable antimicrobial properties. Since some honeys were found to be contaminated with aerobic bacteria or fungi, non-sterile honeys may not be suitable for wound treatment. Further assessment of gamma-irradiated honeys from the best performing honeys would be useful.

Serum cytokine profiles in Behçet's disease: is there a role for IL-15 in pathogenesis?

OBJECTIVES: Behçet's disease (BD) is a multisystem inflammatory disease characterised by recurrent orogenital ulceration, ocular inflammation and skin lesions whose aetiology is currently unknown. We hypothesized that levels of cytokines in the serum might provide either diagnostic or activity markers for the disease. METHODS: Levels of 10 cytokines were analysed in a multiplex bead analysis system as well as IL-15 by ELISA, in 79 serum samples from 52 patients with BD. The same cytokines were also measured in serum samples from 20 patients with recurrent aphthous stomatitis (RAS), as disease controls, and 15 healthy volunteers. The results were correlated with disease activity and current drug therapy. RESULTS: CXCL8 and TNF were the most abundant cytokines and were significantly raised compared to both patients with RAS and healthy controls. IL-15 was present in all samples and was significantly raised in both patients with BD and RAS compared to healthy controls. By comparison, cytokines associated with an adaptive immune response such as IFNgamma and IL-2 were found in few samples, while IL-4 and IL-10 were not detected in any sample. Levels of cytokines correlated with each other suggesting a response to the same stimulus, however, there was no association with either disease activity or treatment. CONCLUSION: Cytokines related to activity of the innate immune response were most prominent in this study and showed good correlation with each other. In particular, it was shown that IL-15 was raised in BD. However, there was no pattern of cytokine expression relating to disease activity or treatment.

Tuberculous orbital apex syndromes.

AIMS: To describe the clinical and radiological features of orbital apex syndromes caused by presumed tuberculosis (TB). METHODS: A review of the discharge summaries of all patients seen in the Medical Eye Unit (MEU), St Thomas' Hospital between 1975 and 2006 identified seven patients with a diagnosis of orbital apex syndrome or optic neuropathy attributable to TB. Case notes and radiology were reviewed retrospectively for each patient. RESULTS: All of the patients were born outside the UK and were HIV-negative. Four presented during 2005-6. Six of the seven patients presented with a visual acuity (VA) of less than counting fingers (CF), but all achieved a VA of 6/9 or better after a median 3 weeks of treatment with antituberculous treatment and systemic corticosteroids. All patients had constitutional symptoms. Chest x ray and CSF were normal in each case, but MRI was abnormal in six. CONCLUSIONS: We report a recent cluster of tuberculous orbital apex syndromes, in the context of an increasing incidence of TB in the UK. The condition is difficult to diagnose, but the combination of high-dose corticosteroids and anti-TB medication was rapidly effective and achieved a good outcome in all cases.

Hypotension and anaemia--a blinding combination.

We present a case of complete blindness following severe dengue haemorrhagic fever complicated by anaemia and a dialysis-related episode of profound hypotension. The clinical and radiological features indicated an optic neuropathy, most likely ischaemic in aetiology. The features of posterior ischaemic optic neuropathy and differential diagnosis are discussed.

Earlier markers for cerebral palsy and clinical research in premature infants.

The diagnosis of cerebral palsy is based on evidence of impairment of the motor system, but symptoms become evident only as a premature infant matures. The diagnosis is made typically at 18 to 24 months of age, corrected for gestational age at birth. An earlier and more accurate way to identify infants destined to develop cerebral palsy may help improve the prognosis for this vulnerable population. For now, no antenatal, perinatal or postnatal test can predict cerebral palsy with a degree of certainty high enough to help providers or parents plan for an infant's future or make the best use of early intervention resources.

Systematic review of ethnic variation in the phenotype of Behcet's disease.

OBJECTIVE: To systematically review the strength of evidence that supports the premise that there are different expressions in the clinical phenotype of Behcet's disease (BD) in different ethnic groups. METHODS: The hierarchy of evidence and inclusion criteria were decided prior to the search for relevant literature. We searched Medline and Embase databases between 1966 and March 2005 for publications related to epidemiology of Behcet's disease or syndrome. Relevant papers were extracted in hard copy and the references of all these papers were then hand searched for further articles. RESULTS: Four population-based studies, of which two were from Turkey, and seven non-population-based comparative studies were found. The majority of literature identified were case series and were not included in the review. There were insufficient cross-sectional studies from different populations to be able to compare phenotypic differences. None of the comparative studies found evidence of a difference in the clinical expression of BD between ethnic groups. CONCLUSIONS: There is insufficient evidence to support the premise that there are different expressions in the clinical phenotype of BD in different ethnic groups. Population-based, cross-sectional surveys or case-control studies using standardized criteria and clear ethnic definitions are suggested to investigate this hypothesis further.

Unilateral electronegative ERG of non-vascular aetiology.

BACKGROUND: Full field and pattern electroretinograms (ERG, PERG) are performed to assess generalised retinal function and macular function, respectively. An (electro) negative full field ERG usually describes an ISCEV standard maximal response in which the b-wave is smaller than a normal or minimally reduced a-wave and indicates dysfunction that is post-phototransduction. The most common cause of a unilateral negative ERG is central retinal artery occlusion (CRAO) or birdshot chorioretinopathy (BCR). This study examines the clinical and electrophysiological features of patients with unilateral negative ERG who do not have CRAO or BCR. METHODS: 12 patients were ascertained with a unilateral negative ERG in whom a vascular aetiology and BCR were excluded. Most presented with symptoms of central retinal dysfunction. In 11 of the 12 patients additional long duration photopic stimuli were used to test cone system ON and OFF responses. RESULTS: All 12 patients had unilateral electronegative bright flash full field ERGs indicating total or relative preservation of rod photoreceptor function, but dysfunction post-phototransduction. Seven of these patients had non-specific inflammatory changes in the eye with the negative ERG. Six patients, including five with inflammatory signs, had involvement of the cone ON response with complete preservation of cone OFF responses. A further three patients showed evidence of cone ON response abnormality with less severe OFF response involvement. CONCLUSION: The ERGs in this heterogeneous group of patients predominantly showed post-phototransduction involvement of the ON pathways. Sparing of the cone OFF response was often observed. The majority of patients had signs of previous inflammation and it is speculated that these highly unusual unilateral changes may be mediated via an autoimmune mechanism.

Intravitreal triamcinalone acetonide for refractory uveitic cystoid macular oedema: longterm management and outcome.

PURPOSE: To establish the role of intravitreal triamcinalone acetonide (TA) in the long term management of refractory uveitic cystoid macular oedema (CMO) and to determine the long term visual outcome in these patients. METHOD: This is a retrospective observational case series. All patients had resistant CMO and active inflammation. The primary outcome measure was complete resolution of CMO on ocular coherence tomography. Visual acuity and intraocular pressure were also monitored. Twelve eyes of twelve patients received 2 (n = 10) to 4 (n = 2) mg of intravitreal TA. All had previously been unresponsive to orbital floor steroids. Fluorescein angiography was performed where indicated. RESULTS: There was complete resolution of CMO in all patients. Nine patients had improvement in acuity at there final follow up (mean follow up 40.5 months). Seven had improved by 2 lines (58%). In 3 patients there was no visual improvement. These 3 failed to respond to re-treatment. Five other patients were re-treated because of recurring CMO and deteriorating VA (median time to re-treatment 4 mths). The mean number of re-treatments for this group with orbital floor TA was three. 41.6% of patients developed ocular hypertension (33.8 mhg mean). One required a trabeculectomy. CONCLUSIONS: 2 mg of intravitreal TA is effective even in cases of resistant uveitic CMO. Although re-treatment is often required, the initial response to treatment can be maintained by subsequent orbital floor steroid injections.

Associations of major histocompatibility complex class I chain-related molecule polymorphisms with Behcet's disease in Caucasian patients.

HLA-B*51 is known to be associated with Behcet's disease (BD) in many ethnic groups. The pathogenic gene, however, may lie close to the HLA-B locus and therefore be in linkage disequilibrium with HLA-B*51. On the basis of the proximity of MIC genes to HLA-B, their expression pattern and their affinity for the activating NKG2D receptor on natural killer (NK) cells and gammadelta T cells, these molecules have been postulated as susceptibility factors in BD. DNA from 56 western European Caucasians with BD and 90 Caucasian controls were analysed by polymerase chain reaction using allele-specific primers for MICA and MICB alleles. An increased allele frequency of MICA*009 was found in the BD patient group (25.0%) when compared with the controls (7.2%). This was associated with a corresponding decrease in MICA*008 in the BD patients (36.6%) compared with the controls (46.7%), which was not significant. MICA*009 was strongly associated with the presence of HLA-B*51 in patients and controls. No significant difference in frequency of MICB alleles was found between patients and controls. Both HLA-B*51 and MICA*009 are strongly associated with BD in a pure Caucasian BD patient group, and the two alleles are in linkage disequilibrium. No MICB allele was found to associate significantly with the disease, an unexpected finding considering the close proximity of the MICA and MICB loci. Our results suggest that while MICB does not influence the development of BD, polymorphisms in MICA may be pathogenic, perhaps through the interaction with NK and gammadelta T cells.