Genotypes of Staphylococcus aureus: On-farm epidemiology and the consequences for prevention of intramammary infections.

Staphylococcus aureus is a highly contagious mastitis-causing pathogen infecting dairy cattle worldwide. Previous studies have shown the presence of different genotypes (GT) on farms. In Switzerland, Staph. aureus genotype B (GTB) is contagious, whereas GTC and other genotypes cause sporadic, noncontagious mastitis. In this study, we evaluated the epidemiological properties of Staph. aureus, together with its genotypes and spa types, on Swiss dairy farms. A total of 21 dairy farms were sampled throughout Switzerland; 10 farms were positive for the contagious Staph. aureus GTB and 11 farms were negative for GTB. Samples were taken from milk, body surfaces of dairy cattle and other animals, milkers, milking equipment, and environmental sites (e.g., parlor, washing room, stall floor, manger, and bedding). The epidemiology of Staph. aureus depended markedly on the genotype. Staphylococcus aureus GTB was associated with mammary gland, intramammary infections (IMI), and milking clusters, whereas GTC and other genotypes were related to cow and other animal surfaces and occasionally to environment. Genotype C was by far the most common subtype in cattle and was found on GTB-negative and GTB-positive farms. Each farm had a predominant genotype, such as GTB, GTC, GTA, or GTF, but a few farms were almost free from Staph. aureus. The genotypes and spa types of Staph. aureus detected in the noses of milkers clearly differed from those found in dairy cattle, other animals, milking equipment, and the environment. Exceptions were GTS (spa type t034) and GTF (t899), which crossed the species barrier. In most cases, however, the species barrier was maintained because Staph. aureus is adapted to a particular host and even to particular body sites. As biological properties differ among the genotypes, new guidelines to prevent IMI caused by different genotypes were established: classical measures to prevent IMI caused by contagious pathogens still hold for GTB but not for Staph. aureus genotypes that are opportunistic colonizers of bovine skin (e.g., GTC and GTA). For those genotypes, protection of the skin from minor lesions and wounds, particularly on the hocks, is essential.

Human Mozdok leptospirosis first diagnosed by serum agglutinin-absorption tests in the Slovak Republic.

INTRODUCTION: The serovar Mozdok related leptospirosis in humans were not yet feasibly diagnosed using merely the standard micro-agglutination test (MAT) what was perhaps due to the impossibility to distinguish them from illnesses that are caused by Leptospira strains belonging to other serovars of the serogroup Pomona. On the contrary, leptospires of the Mozdok serovar were cultured from rodents and domestic animals world-wide including Central Europe where only Leptospira strains of the serovars Pomona and Mozdok are known to be present till now. STUDY OBJECTIVE: The aim of the study was to discover if leptospires of Mozdok serovar may cause human leptospirosis that remained hidden till now among infections diagnosed merely by MAT as Pomona illnesses. MATERIAL AND METHODS: The reference Leptospira strains of Pomona and Mozdok serovars (Pomona and 5621), as well as three endemic, and in some tests only two strains of human and pig origin (Simon, S-23, Pöstényi), and two strains of rodent provenance - Apodemus agrarius (M-210/98 and M-71/01) were used for this purpose. First, the endemic strains were assigned to one of the afore-mentioned two serovars by agglutinin cross-absorption tests performed using rabbit immune sera, monoclonal antibodies and random amplified polymorphic DNA methods. Afterwards, twenty-one sera of patients with a Pomona leptospirosis confirmed by MAT were examined by agglutinin absorption test (AAT). RESULTS: Based on the results of the mentioned laboratory method used, the endemic Leptospira strains of human and pig origin could be affiliated to the serovar Pomona, while those of rodent origin were classified as serovar Mozdok strains. Out of the 21 patients sera, an illness caused by the serovar Mozdok strains was found out in 13 cases and a disease caused by serovar Pomona strains in 8 cases. Their differentiation was made on the strength of the following results of AATs: All strains from the serovar Mozdok have completely absorbed antibodies (anti-Pomona and anti-Mozdok) from the tested sera, however following the absorption of these sera with the Pomona strains, high levels of residual antibodies reacting in MAT with the Mozdok strains have still persisted. In this way, it was possible to prove the Mozdok infection in thirteen patients. On the contrary, following the absorption of the sera with the strains of the serovar Pomona, a complete absorption of all antibodies (anti-Pomona and anti-Mozdok) was achieved in seven cases using the strain Simon, and in one case with the strain S-23, whereas after absorption using the Pomona strain, the residual antibodies were still present in all sera, and also in the majority of them when they were absorbed using the strains S-23 and Pöstényi. In this context, the Pomona infection was determined in the case of eight patients. Hence it follows that not all strains of the Pomona serovar were suitable for the AATs. CONCLUSION: The presence of the human Mozdok leptospirosis was confirmed for the first time by the use of the agglutinin absorption test. A clear correlation between the habitat areas of the A. agrarius and the patients who were infected with the strains of the Mozdok serovar was determined.

DNA relatedness and serotyping of Leptospira strains.

BACKGROUND: In a previous study (5), the constructed phylogenetic tree for leptospires belonging to 12 different serovars common in Central Europe made the prediction of serovar from knowing the genotype and vice versa possible. OBJECTIVE: The study was aimed at investigation of the usefulness of such procedure to distinguish in between at present to us available and worldwide accepted reference strains of pathogenic Leptospira serovars. MATERIAL AND METHODS: One hundred and seventy seven Leptospira strains representing different serovars were tested. DNA fingerprints of these strains were performed, digitally captured and as described earlier of those phylogenetic tree using different fingerprinting software was constructed using UPGMA clustering method with band matching by the Dice coefficient (5). RESULTS: At this tree, 145 of 177 Leptospira strains tested each took a unique position, and the remaining 32 strains were distributed at 15 different positions (each of 14 positions taken by two different strains and one position taken by four strains). CONCLUSION: The constructed phylogenetic tree likely to be very useful in prediction of Leptospira serovar in most cases of an infection so the saving time and being helpful in serovar identification of the pathogenic agent (Fig. 1, Ref. 9).

Personalized bacteriophage therapy to treat pandrug-resistant spinal Pseudomonas aeruginosa infection.

Bone and joint infections (BJI) are one of the most difficult-to-treat bacterial infection, especially in the era of antimicrobial resistance. Lytic bacteriophages (phages for short) are natural viruses that can selectively target and kill bacteria. They are considered to have a high therapeutic potential for the treatment of severe bacterial infections and especially BJI, as they also target biofilms. Here we report on the management of a patient with a pandrug-resistant Pseudomonas aeruginosa spinal abscess who was treated with surgery and a personalized combination of phage therapy that was added to antibiotics. As the infecting P. aeruginosa strain was resistant to the phages developed by private companies that were contacted, we set up a unique European academic collaboration to find, produce and administer a personalized phage cocktail to the patient in due time. After two surgeries, despite bacterial persistence with expression of small colony variants, the patient healed with local and intravenous injections of purified phages as adjuvant therapy.

Unravelling the structure of the lamellipodium.

Summary Pushing at the cell front is the business of lamellipodia and understanding how lamellipodia function requires knowledge of their structural organization. Analysis of extracted, critical-point-dried cells by electron microscopy has led to a current dogma that the lamellipodium pushes as a branched array of actin filaments, with a branching angle of 70 degrees , defined by the Arp2/3 complex. Comparison of different preparative methods indicates that the critical-point-drying-replica technique introduces distortions into actin networks, such that crossing filaments may appear branched. After negative staining and from preliminary studies by cryo-electron tomography, no clear evidence could be found for actin filament branching in lamellipodia. From recent observations of a sub-class of actin speckles in lamellipodia that exhibit a dynamic behaviour similar to speckles in the lamella region behind, it has been proposed that the lamellipodium surfs on top of the lamella. Negative stain electron microscopy and cryo-electron microscopy of fixed cells, which reveal the entire complement of filaments in lamellipodia show, however, that there is no separate, second array of filaments beneath the lamellipodium network. From present data, we conclude that the lamellipodium is a distinct protrusive entity composed of a network of primarily unbranched actin filaments. Cryo-electron tomography of snap-frozen intact cells will be required to finally clarify the three-dimensional arrangement of actin filaments in lamellipodia in vivo.

Diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH) and multiple pulmonary epithelioid hemangioendothelioma (PEH): a case report.

We report a case of a 76-year-old female with multiple lung nodules (Fig. 1 Rx). Pathologic evaluation of the lower left video-assisted thoracoscopic surgery (VATS) lobectomy VATS-lobectomy showed four nodules that were described as pulmonary epithelioid hemangio-endothelioma (PEH); the immunohistochemical stains showed that the neoplastic cells expressed CD31, a variable expression for factor VIII and a low expression of CD34. In the remaining parenchyma of the lobe, multiple nests of neuroendocrine cells were observed with immunohistochemical confirmation, and the diagnosis was diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH). To our knowledge, the association between PEH and DIPNECH has never been described in the literature.

The effect of neoplasia-induced hypercalcemia on the neuroendocrine axis of the Fischer rat.

The effects of neoplasia-induced hypercalcemia on the hypothalamic-pituitary axis of the Fischer rat were determined by measuring the responses of TSH to TRH, PRL to TRH and haloperidol, and LH to LHRH in 3 groups of 8 rats prior to and at 4 and 8 days after transplantation of a Leydig cell tumor. The mean serum calcium was 8.2 +/- 0.2 mg/dl in 8 Fischer rats pretransplantation; had risen at 4 days posttransplantation to 9.8 +/- 0.2 mg/dl; and at 8 days was 11.2 +/- 0.1 mg/dl. TSH response to TRH was greater in the pretransplant rats than in the groups studied at 4 and 8 days posttransplant. Basal PRL levels and PRL responses to TRH and haloperidol were less in the groups studied at 4 and 8 days posttransplant. Basal LH values were similar in all 3 groups, but the LH response to LHRH was less for the posttransplant groups. The TSH response to TRH, PRL responses to TRH and haloperidol, and LH responses to LHRH were less in the 8 days posttransplants than in the groups studied at 4 days posttransplantation. There were significant negative correlations between TSH (r = -0.79) and PRL (r = -0.80) responses to TRH, PRL responses (r = -0.94) to haloperidol and LH responses (r = -0.91) to LHRH and the serum calcium. The results indicate that increasing levels of serum calcium following Leydig cell tumor transplantation are associated with suppression of adenohypophysial release of TSH, PRL, and LH; the degree of suppression is related to the magnitude of hypercalcemia achieved in this model.

Glycyl-L-glutamine antagonizes alpha-MSH-elicited thermogenesis.

The objective of this study was to determine whether glycyl-L-glutamine [beta-endorphin(30-31)] modulates the thermoregulatory actions of alpha-MSH. Microinjection of alpha-MSH (0.06 nmol) into PGE2-responsive thermogenic sites in the medial preoptic area of rats generated a hyperthermic response, inducing a 0.85 +/- 0.19 degrees C rise in colonic temperature (Tc) within 45 min. Coadministration of glycyl-L-glutamine (3.0 nmol) completely blocked the response, maintaining Tc at baseline levels. This was not attributable to glycyl-L-glutamine hydrolysis because coadministration of glycine and glutamine had no effect on alpha-MSH-induced thermogenesis. Glycyl-L-glutamine, injected alone, was similarly without effect. These data indicate that glycyl-L-glutamine inhibits alpha-MSH-induced thermogenesis but is devoid of thermoregulatory activity itself.

Effects of central alpha-MSH injections on performance in a cued discrimination task.

Alpha-MSH has been implicated in changing attention behavior following peripheral injections, but no brain sites were studied. In the present report, alpha-MSH was injected directly into specific sites in the medial anterior hypothalamic/preoptic area (MAHPOA) while measuring performance in a visually cued discrimination task. Alpha-MSH injections resulted in reduced errors, indicated by decreased responding during noncued intervals, but no change in responding to correct cues. The improved error rate was consistent with attentional changes in a variety of paradigms. Attentional and motivational parameters were differentiated. The injected alpha-MSH appears to act on an inhibitory component of an attentional mechanism.

Inhibition of interleukin-1beta and prostaglandin E(2) thermogenesis by glycyl-glutamine, a pro-opiomelanocortin-derived peptide.

Interleukin-1beta (IL-1beta) and other cytokines produce fever by stimulating prostaglandin E(2) (PGE(2)) synthesis in thermoregulatory regions of the preoptic area and anterior hypothalamus (POA/AH). Prostaglandin E(2) is thought to raise body temperature, at least in part, by stimulating beta-endorphin release from pro-opiomelanocortin neurons that innervate the POA/AH. In this study, we investigated whether glycyl-glutamine (beta-endorphin(30-31)), an inhibitory dipeptide synthesized from beta-endorphin post-translationally, inhibits IL-1beta and PGE(2)-induced hyperthermia. Hyperthermic sites were identified by microinjecting PGE(2) (3 fmol/1 microl) into the medial preoptic area (mPOA) of conscious, unrestrained rats. Interleukin-1beta (1 U) injection into the same PGE(2) responsive thermogenic sites in the mPOA elicited a prolonged rise in colonic temperature (T(c)) (+1.02+/-0.06 degrees C) that persisted for at least 2 h. Glycyl-glutamine (3 nmol) co-injection into the mPOA inhibited IL-1beta thermogenesis completely (T(c)=-0.18+/-0.22 degrees C). Glycyl-glutamine had no effect on body temperature when given alone to normothermic rats. Co-injection of individual amino acids, glycine and glutamine (3 nmol each amino acid), failed to influence IL-1beta-induced thermogenesis, which indicates that Gly-Gln hydrolysis does not explain its inhibitory activity. Glycyl-glutamine (3 nmol) also prevented the rise in body temperature produced by PGE(2) (PGE(2)=0.89+/-0.05 degrees C; PGE(2) plus Gly-Gln=-0.16+/-0.14 degrees C), consistent with evidence that PGE(2) mediates IL-1beta-induced fever. These findings demonstrate that Gly-Gln inhibits the thermogenic response to endogenous pyrogens.

Organization of central hyperthermic mechanisms in helium-cold hypothermic hamsters.

We have investigated the ability of three hyperthermic stimuli (PGE2, 5-HT and ACh) to elicit hyperthermia in the Helium-Cold (He-Cold) hypothermic hamster. Hamsters in these conditions are poikilothermic and will passively follow room temperature in a regulated cold room. Animals were injected centrally at AH/POA sites via an indwelling guide tube at body temperatures maintained between 9-12 degrees C. Active sites in the AH/POA were determined prior to the experiment by PGE2 injection. PGE2 injection at an effective AH/POA site immediately reversed the anesthetic induced hypothermia in warm air. Hamsters were induced into hypothermia by the He-Cold induction method and body temperatures were maintained in a 9 degrees C cold room. Colonic temperatures were monitored at 5 minute intervals by a YSI thermistor probe and telethermometer. Central injections of 5-HT (2 micrograms/microliter) and ACh (50 micrograms/microliter) at effective AH/POA sites evoked significant increases in colonic temperature in He-Cold hamsters. PGE2 injections were not different from saline control injections and did not elicit pronounced temperature changes in these animals. Specific blockade of the 5-HT and ACh temperature increases was demonstrated with specific antagonist injections. The results suggest that the central organization of heat-gain mechanisms in the AH/POA is the same as normothermic animals even at temperatures well below those previously investigated.

ACh and 5-HT stimulated thermogenesis at different core temperatures in the He-Cold hypothermic hamster.

Hamsters in deep experimentally induced hypothermia, at body temperatures between 7 degrees C and 11.5 degrees C, were microinjected with 5-HT and ACh at brain sites in the anterior-preoptic area of the hypothalamus (AH/POA). ACh or 5-HT was injected into an AH/POA site at different starting core temperatures in different groups of hypothermic hamsters. Colonic temperatures (Tc) were maintained, following He-Cold induction, in a temperature controlled environmental chamber and measured with a YSI thermister probe and YSI telethermometer. Injections of either 5-HT or ACh at Tc's between 7.0 degrees C and 9.0 degrees C elicited only modest increases in Tc i.e., 0.3 degrees C--0.6 degrees C, respectively. As Tc increased, however, to ranges between 9.1 degrees C--10.0 degrees C and in different animals to greater than 10 degrees C both ACh and 5-HT at the same sites elicited significant increases in Tc, 1.5 degrees C for 5-HT and 2.2 degrees C for ACh compared to saline injections. These data suggest that at the lowest Tc's we are observing a "cold block" of temperature sensitive sites in the AH/POA. Increasing the starting Tc beyond 9.0 degrees C however, evokes significant increases in heat-gain following AH/POA injection of either ACh or 5-HT. These data are consistent with Myers' observations concerning the organization of heat-gain mechanisms at AH/POA sites. In addition, they suggest that both the afferent limb of the heat-gain circuit (5-HT) and the efferent limb of the circuit (ACh) are functionally impaired when Tc is close to the physiological limit in the He-Cold hypothermic hamster.

Recovery by push-pull perfusion of neurochemicals released within the cuneate nucleus of the cat by somatosensory stimulation.

The present work describes a combination of techniques for the identification of neurochemicals released within the cuneate nucleus. During electrical stimulation of the superficial radial nerve, the extracellular fluid of the nucleus is continuously sampled by push-pull perfusion. In addition, the population electrical activity of peripheral nerve as well as the activity of cuneate neurons are recorded. Subsequently, the neurochemical content of the sampled fluid is assessed by HPLC analysis. The comparison of sampled fluid content during control (no stimulation) versus stimulation runs indicates that somatosensory stimulation elicits the release of specific neurochemicals within the cuneate nucleus. The possible sources of released neurochemicals are discussed.

Glycyl-L-glutamine injected centrally suppresses alcohol drinking in P rats.

Recent reports show that central beta-endorphin (1-31) injection augments the volitional intake of alcohol. Correspondingly, alcohol drinking stimulates beta-endorphin (1-31) release from the hypothalamus of the rat. Glycyl-l-glutamine (Gly-Gln) is produced in beta-endorphin-containing neurons and is co-released with beta-endorphin(1-31) and other processing products. Because Gly-Gln is apparently an endogenous antagonist of beta-endorphin(1-31) in several systems, the present study was designed to investigate the hypothesis that Gly-Gln injected i.c.v. would alter voluntary alcohol drinking in the genetic, high-alcohol-preferring P rat. After a guide tube was implanted stereotaxically above the lateral cerebral ventricle, the rats were offered 3-30% alcohol over 10 days, and then given their maximally preferred concentration of alcohol in the presence of water for the remainder of the experiment. Gly-Gln or artificial cerebrospinal fluid (CSF) vehicle then was injected i.c.v. in a dose of 10 or 100 nmol for 3 consecutive days, which was followed by a 7-day postinjection interval. Gly-Gln suppressed significantly the intakes of alcohol in terms of both g/kg and proportion to total fluid. During the postinjection days, alcohol drinking continued to be suppressed, whereas neither the daily intakes of food or water nor the body weights of the rats were changed. The present results are consistent with the concept of a functional antagonism by Gly-Gln of the role of beta-endorphin(1-31) in mediating certain central functions. These results demonstrate that alcohol consumption is suppressed by the direct intracerebral application of this unique peptide.

EMCD real space maps of Magnetospirillum magnetotacticum.

In this study we combine energy loss magnetic circular dichroism (EMCD) and energy filtered transmission electron microscopy (EFTEM) to map magnetic properties of nanoparticles. We show that it is a functional tool for investigating the magnetic behaviour of bio-mineralized magnetite crystals of Magnetospirillum magnetotacticum. We find that the spatial resolution of our experimental set-up is in the range of less than 2 nm. The results are compared with EMCD studies of abiogenic magnetite.

The Nationwide Austrian Aspergillus Registry: a prospective data collection on epidemiology, therapy and outcome of invasive mould infections in immunocompromised and/or immunosuppressed patients.

A prospective, observational, multicentre study was performed to assess the incidence, diagnosis, epidemiology and outcome of invasive mould infections (IMIs) reported to the Nationwide Austrian Aspergillus Registry. In total, 186 cases were recorded, corresponding to an annual incidence of 42 cases/1000 patients at risk or 2.36 cases/100000 inhabitants. Patients with acute myelogenous leukaemia (34%) and lung transplant recipients (17%) are currently at highest risk for IMI, followed by a mixed population with impaired immunity (14%). In total, 34%, 30% and 36% were proven, probable and possible cases of IMI. Predominant pathogens were Aspergillus spp. (67%), followed by the zygomycetes (28%). Voriconazole was the most frequently administered agent (38%), followed by caspofungin (20%) and posaconazole (19%). Eighty patients (43%) received antifungal prophylaxis for ≥7 days, 30% of whom (24 patients) suffered from a breakthrough infection. The overall crude 12-week mortality was 34%. Multivariate analysis showed that outcome and survival did not correlate with the status of fungal disease, breakthrough infection, fungal species or age (P>0.05). Aspergillosis remains the most commonly identified IMI amongst immunocompromised and/or immunosuppressed patients, but other moulds constitute a significant problem. Survival from IMIs appears to have improved and the main challenge is to overcome breakthrough fungal infections.

Utility of phylogenetic studies in the identification of Leptospira strains.

A phylogenetic tree, which distinguishes between the serovars and serogroups of leptospires common in Central Europe was constructed using an established RAPD procedure together with digital reading and evaluation (using different computer software programs) of the generated amplified DNA patterns. The application of this procedure has revealed a consistent correspondence between serogroup and genotype (position in constructed tree) in 69 cases, and serovar and genotype in 72 cases, of wild strains of leptospires. There was an agreement between serovar and genotype in cases of strains of Grippotyphosa, Pomona, Mozdok, Arborea and Sorexjalna as well as between serogroup and genotype in cases of Australis, Bataviae and Sejroe. With the procedure used in this study, it was not possible to distinguish between reference strains of serovars Jalna, Bratislava and Lora (all serogroup Australis) as well as between serovars Icterohaemorrhagiae and Copenhageni (both of serogroup Icterohaemorrhagiae). In contrast to this, wild strains belonging to serogroup Sejroe were distributed between Polonica, Istrica, Saxkoebing and Sejroe serovars. Endemic strains of leptospires tested, were also distinguishable.

Adjunctive homeopathic treatment in patients with severe sepsis: a randomized, double-blind, placebo-controlled trial in an intensive care unit.

BACKGROUND: Mortality in patients with severe sepsis remains high despite the development of several therapeutic strategies. The aim of this randomized, double-blind, placebo-controlled trial was to evaluate whether homeopathy is able to influence long-term outcome in critically ill patients suffering from severe sepsis. METHODS: Seventy patients with severe sepsis received homeopathic treatment (n = 35) or placebo (n = 35). Five globules in a potency of 200c were given at 12h interval during the stay at the intensive care unit. Survival after a 30 and 180 days was recorded. RESULTS: Three patients (2 homeopathy, 1 placebo) were excluded from the analyses because of incomplete data. All these patients survived. Baseline characteristics including age, sex, BMI, prior conditions, APACHE II score, signs of sepsis, number of organ failures, need for mechanical ventilation, need for vasopressors or veno-venous hemofiltration, and laboratory parameters were not significantly different between groups. On day 30, there was non-statistically significantly trend of survival in favour of homeopathy (verum 81.8%, placebo 67.7%, P= 0.19). On day 180, survival was statistically significantly higher with verum homeopathy (75.8% vs 50.0%, P = 0.043). No adverse effects were observed. CONCLUSIONS: Our data suggest that homeopathic treatment may be a useful additional therapeutic measure with a long-term benefit for severely septic patients admitted to the intensive care unit. A constraint to wider application of this method is the limited number of trained homeopaths.