Evaluation of the Efficacy of Doxycycline, Ciprofloxacin, Levofloxacin, and Co-trimoxazole Using In Vitro and In Vivo Models of Q Fever.

Q fever, caused by the intracellular pathogen Coxiella burnetii, is traditionally treated using tetracycline antibiotics, such as doxycycline. Doxycycline is often poorly tolerated, and antibiotic-resistant strains have been isolated. In this study, we have evaluated a panel of antibiotics (doxycycline, ciprofloxacin, levofloxacin, and co-trimoxazole) against C. burnetii using in vitro methods (determination of MIC using liquid and solid media; efficacy assessment in a THP cell infection model) and in vivo methods (wax moth larvae and mouse models of infection). In addition, the schedule for antibiotic treatment has been evaluated, with therapy initiated at 24 h pre- or postchallenge. Both doxycycline and levofloxacin limited overt clinical signs during treatment in the AJ mouse model of aerosol infection, but further studies are required to investigate the possibility of disease relapse or incomplete bacterial clearance after the antibiotics are stopped. Levofloxacin was well tolerated and therefore warrants further investigation as an alternative to the current recommended treatment with doxycycline.

Dose-dependant acute or subacute disease caused by Burkholderia pseudomallei strain NCTC 13392 in a BALB/c aerosol model of infection.

AIMS: The goal of this study was to examine, for the first time, the virulence and pathogenicity of aerosolized Burkholderia pseudomallei, strain NCTC 13392, in BALB/c mice in order to develop an animal model for testing novel medical countermeasures (MCMs) for the treatment of human acute and subacute (a disease state between acute and chronic) melioidosis. METHODS AND RESULTS: BALB/c mice were exposed to varying doses of aerosolized bacteria. Acute disease was seen in animals exposed to a very-high dose (≥103  CFU per animal) and death occurred 3-4 days postchallenge (pc). Bacteria were detected in the lungs, liver, kidney and spleen. In contrast, animals exposed to a low dose (<10 CFU per animal) survived to the end of the study (day 30 pc) but developed weight loss, a bacterial tissue burden and increasing clinical signs of infection from day 20 pc onwards, mimicking a subacute form of the disease. Pathological changes in the tissues mirrored these findings. CONCLUSIONS: This proof of concept study has shown that B. pseudomallei strain NCTC 13392 is virulent and pathogenic in BALB/c mice, when delivered by aerosol. By varying the doses of aerosolized bacteria it was possible to mimic characteristics of both human acute and subacute melioidosis, at the same time, within the same study. SIGNIFICANCE AND IMPACT OF THE STUDY: Burkholderia pseudomallei, the aetiological agent of melioidosis, causes a serious and often fatal disease in humans and animals. Novel MCMs are urgently needed for both public health and biodefense purposes. The present model provides a useful tool for the assessment and evaluation of new MCMs (e.g. therapeutics and vaccines) and offers the potential for testing new treatments for both subacute to chronic and acute melioidosis prior to human clinical trials.

Sequence of pathogenic events in cynomolgus macaques infected with aerosolized monkeypox virus.

UNLABELLED: To evaluate new vaccines when human efficacy studies are not possible, the FDA's "Animal Rule" requires well-characterized models of infection. Thus, in the present study, the early pathogenic events of monkeypox infection in nonhuman primates, a surrogate for variola virus infection, were characterized. Cynomolgus macaques were exposed to aerosolized monkeypox virus (10(5) PFU). Clinical observations, viral loads, immune responses, and pathological changes were examined on days 2, 4, 6, 8, 10, and 12 postchallenge. Viral DNA (vDNA) was detected in the lungs on day 2 postchallenge, and viral antigen was detected, by immunostaining, in the epithelium of bronchi, bronchioles, and alveolar walls. Lesions comprised rare foci of dysplastic and sloughed cells in respiratory bronchioles. By day 4, vDNA was detected in the throat, tonsil, and spleen, and monkeypox antigen was detected in the lung, hilar and submandibular lymph nodes, spleen, and colon. Lung lesions comprised focal epithelial necrosis and inflammation. Body temperature peaked on day 6, pox lesions appeared on the skin, and lesions, with positive immunostaining, were present in the lung, tonsil, spleen, lymph nodes, and colon. By day 8, vDNA was present in 9/13 tissues. Blood concentrations of interleukin 1ra (IL-1ra), IL-6, and gamma interferon (IFN-γ) increased markedly. By day 10, circulating IgG antibody concentrations increased, and on day 12, animals showed early signs of recovery. These results define early events occurring in an inhalational macaque monkeypox infection model, supporting its use as a surrogate model for human smallpox. IMPORTANCE: Bioterrorism poses a major threat to public health, as the deliberate release of infectious agents, such smallpox or a related virus, monkeypox, would have catastrophic consequences. The development and testing of new medical countermeasures, e.g., vaccines, are thus priorities; however, tests for efficacy in humans cannot be performed because it would be unethical and field trials are not feasible. To overcome this, the FDA may grant marketing approval of a new product based upon the "Animal Rule," in which interventions are tested for efficacy in well-characterized animal models. Monkeypox virus infection of nonhuman primates (NHPs) presents a potential surrogate disease model for smallpox. Previously, the later stages of monkeypox infection were defined, but the early course of infection remains unstudied. Here, the early pathogenic events of inhalational monkeypox infection in NHPs were characterized, and the results support the use of this surrogate model for testing human smallpox interventions.

Efficacy of liposome-encapsulated ciprofloxacin in a murine model of Q fever.

Encapsulation of antibiotics may improve treatment of intracellular infections by prolonging antibiotic release and improving antibiotic uptake into cells. In this study, liposome-encapsulated ciprofloxacin for inhalation (CFI) was evaluated as a postexposure therapeutic for the treatment of Coxiella burnetii, the causative agent of Q fever. Intranasal treatment of male A/Jola (A/J) mice with CFI (50 mg/kg of body weight) once daily for 7 days protected mice against weight loss and clinical signs following an aerosol challenge with C. burnetii. In comparison, mice treated twice daily with oral ciprofloxacin or doxycycline (50 mg/kg) or phosphate-buffered saline (PBS) lost 15 to 20% body weight and exhibited ruffled fur, arched backs, and dehydration. Mice were culled at day 14 postchallenge. The weights and bacterial burdens of organs were determined. Mice treated with CFI exhibited reduced splenomegaly and reduced bacterial numbers in the lungs and spleen compared to mice treated with oral ciprofloxacin or doxycycline. When a single dose of CFI was administered, it provided better protection against body weight loss than 7 days of treatment with oral doxycycline, the current antibiotic of choice to treat Q fever. These data suggest that CFI has potential as a superior antibiotic to treat Q fever.

A Crimean-Congo hemorrhagic fever (CCHF) viral vaccine expressing nucleoprotein is immunogenic but fails to confer protection against lethal disease.

Crimean-Congo Hemorrhagic Fever (CCHF) is a severe tick-borne disease, endemic in many countries in Africa, the Middle East, Eastern Europe and Asia. Between 15-70% of reported cases are fatal with no approved vaccine available. In the present study, the attenuated poxvirus vector, Modified Vaccinia virus Ankara, was used to develop a recombinant candidate vaccine expressing the CCHF virus nucleoprotein. Cellular and humoral immunogenicity was confirmed in 2 mouse strains, including type I interferon receptor knockout mice, which are susceptible to CCHF disease. Despite the immune responses generated post-immunisation, the vaccine failed to protect animals from lethal disease in a challenge model.

Alternative BCG delivery strategies improve protection against Mycobacterium tuberculosis in non-human primates: Protection associated with mycobacterial antigen-specific CD4 effector memory T-cell populations.

Intradermal (ID) BCG injection provides incomplete protection against TB in humans and experimental models. Alternative BCG vaccination strategies may improve protection in model species, including rhesus macaques. This study compares the immunogenicity and efficacy of BCG administered by ID and intravenous (IV) injection, or as an intratracheal mucosal boost (ID + IT), against aerosol challenge with Mycobacterium tuberculosis Erdman strain. Disease pathology was significantly reduced, and survival improved, by each BCG vaccination strategy, relative to unvaccinated animals. However, IV induced protection surpassed that achieved by all other routes, providing an opportunity to explore protective immunological mechanisms using antigen-specific IFN-γ ELISpot and polychromatic flow cytometry assays. IFN-γ spot forming units and multifunctional CD4 T-cell frequencies increased significantly following each vaccination regimen and were greatest following IV immunisation. Vaccine-induced multifunctional CD4 T-cells producing IFN-γ and TNF-α were associated with reduced disease pathology following subsequent M.tb challenge; however, high frequencies of this population following M.tb infection correlated with increased pathology. Cytokine producing T-cells primarily occupied the CD4 transitional effector memory phenotype, implicating this population as central to the mycobacterial response, potentially contributing to the stringent control observed in IV vaccinated animals. This study demonstrates the protective efficacy of IV BCG vaccination in rhesus macaques, offering a valuable tool for the interrogation of immunological mechanisms and potential correlates of protection.

Early lesions following aerosol challenge of rhesus macaques (Macaca mulatta) with Mycobacterium tuberculosis (Erdman strain).

Three rhesus macaques (Macaca mulatta) were challenged with Mycobacterium tuberculosis (Mtb), Erdman strain, as part of studies to investigate lesion development at early time points in tuberculosis (TB) and to assess computed tomography (CT) as a method of monitoring disease progression in vivo. Animals were challenged with either a high, mid or low dose of aerosolized Mtb. The low-dose animal was killed humanely at 24 days post challenge (dpc) and the remaining animals at 25 dpc. Abnormalities in clinical parameters were observed in all animals, but clinical signs relating to respiratory disease were not seen. Pulmonary changes consistent with TB infection were detected by CT at 21 dpc and magnetic resonance imaging (MRI) post mortem. Pulmonary nodule counts obtained from both imaging techniques were directly proportional to the challenge dose and correlated with gross and microscopical lesion counts. On gross and microscopical examination, lesions of similar size and morphology were observed in the lungs of all three animals, with the majority containing necrotic foci. Concomitant gross and microscopical, granulomatous lesions were observed in the tracheobronchial lymph nodes of all animals together with evidence of systemic spread. These findings further contribute to our understanding and knowledge of early lesion formation in the lungs of non-human primates.

Comparison of norfloxacin with cotrimoxazole for infection prophylaxis in acute leukemia. The trade-off for reduced gram-negative sepsis.

A total of 63 neutropenic patients receiving cytotoxic therapy for acute leukemia were randomly allocated to receive norfloxacin (400 mg every 12 hours) or cotrimoxazole (160/800 mg every 12 hours) to prevent bacterial infection. Compliance was more than 95 percent and no adverse effects attributable to the study drugs were observed. The overall incidence of febrile illness (67 percent) was similar between the groups; however, no gram-negative bacillary infections were observed in 31 norfloxacin recipients compared with four of 32 cotrimoxazole recipients. Furthermore, nine norfloxacin recipients had 17 gram-positive bacteremias compared with two in two cotrimoxazole recipients (p = 0.0034). Norfloxacin was more effective than cotrimoxazole for preventing acquisition of aerobic gram-negative bacilli in surveillance cultures. Neither study drug allocation nor the presence of an indwelling central venous catheter influenced outcome among the 42 patients who subsequently received empiric systemic antibiotics for suspected infection. Although gram-positive infection remains an unsolved problem, norfloxacin appears to be a safe, effective, well-tolerated alternative to cotrimoxazole for preventing gram-negative infection in neutropenic patients with acute leukemia.

Pecking preferences and pre-dispositions in domestic chicks: implications for the development of environmental enrichment devices.

Environmental enrichment is thought likely to benefit chickens and farmers in many ways; these include reduced fearfulness and feather pecking and improved productivity. Enrichment devices would intuitively be more effective if they reliably attracted and sustained appreciable interest but many fail to do so. This may reflect the fact that the choice of stimuli often reflects availability and human preconceptions rather than a critical consideration of the birds' preferences and pre-dispositions. We had previously identified string as a particularly attractive pecking stimulus for chicks and adult hens (Gallus gallus domesticus) of a laying strain (ISA Brown). In the present study we found that chicks of another laying strain (Lohmann Brown) also pecked sooner and more at a bunch of string than at chains or beads (Experiment 1). White or yellow strings were preferred to red, green or blue ones (Experiment 2) and white string elicited more pecking than did combinations of white and yellow or of all five colours (Experiment 3). Varying the length and width of the bunches of string exerted no detectable effects on pecking (Experiment 4) whereas incorporating small, shiny beads in the white string devices actually reduced pecking (Experiment 5). Virtually all the devices elicited progressively more interest with repeated presentation; this trend was particularly marked for white string. Collectively, the present findings demonstrate that young domestic chicks have clear and specific pecking preferences. Although the magnitude of response varied across experiments, white string consistently elicited the most interest. Our two main conclusions are: (i) white or yellow strings were particularly attractive stimuli that drew increasing interest, at least in the short term, and (ii) simple devices were preferred to more complex ones, or at least to those used here.

Matching, attunement and the psychoanalytic dialogue.

This paper is concerned with unformulated events in analytic sequences before the crystallization that makes verbal interpretation possible. It examines the use of certain concepts from infant research, particularly amodal perception and attunement, which may be of use to the clinical analyst. It is noted that time-sequence concepts, such as musical ones, like affect-attunement, are poorly developed in psychoanalysis. Illustrations of attunements in analysis are attempted by means of doggerel verses about some patients. It is suggested that a progressive movement or dialectic between the pre-verbal affect-attunement level and formalized interpretation may be optimal in analytic therapy.

Infinite experiences, affects and the characteristics of the unconscious.

The work of Matte-Blanco is little understood by psychoanalysts largely because he used mathematical logic as an essential method to elucidate psychoanalytic theory. His findings, however, seem to be important and warrant this simple introductory paper explaining key ideas. Two fundamentally distinct processes, defined as asymmetrical and symmetrical logics are introduced and illustrated. Asymmetrical logic is analogous to secondary process while symmetrical logic has affinity with primary process. Both logic seem to interweave in any thought processes but with asymmetry predominating in scientific thought while symmetrical logic is most evident in psychosis and dreams. It is furthermore argued that the various characteristics of the unconscious, as described by Freud, can be seen as different mental outcomes with symmetrical logic at the base of all of them. A parallel is discovered by Matte-Blanco between the characteristics of symmetrical logic and those of infinite sets as defined mathematically. This leads to an investigation of the concept of infinity and of psychological infinite experiences. These can be readily detected in omnipotence, omniscience, and idealization, but they also seem to occur in extreme emotional states such as being in love, dread and grief. If nuclei of extreme states are contained in any affect, then it is likely that all affects in their cognitive aspects contain experiences of infinity. Symmetrical logic may thus be a common background both to the characteristics of the unconscious and emotionality generally.

Some functions of being fair and just--or not, in clinical psychoanalysis.

The author suggests that the concepts of fairness and justice might be psychoanalytically useful. They are social ideas but often arise clinically. Since psychoanalysis is about intimate and internal events, the wide social and legal settings associated with formal justice are not addressed. Fairness and informal justice arise more intimately and are central here. Fairness is concerned with thorough consideration of an issue. To be fair, the people involved must be considered with impartiality before a decision. Justice generally is more formal, aiming to ensure the integrity of a system, its parts and individual members. It is noted that psychopathogenic conditions can arise in conflicting situations involving fairness and justice. Clinical illustrations suggest that the psychoanalytic process can implicitly aim to resolve their after-effects. Technique is addressed in this light. A central suggestion emerges here. The philosopher Jürgen Habermas proposes that justice rests essentially upon opportunity for argumentation between all those affected. The author suggests that psychoanalytic therapy is likewise a constructive argumentation.

Early lesions following aerosol infection of rhesus macaques (Macaca mulatta) with Mycobacterium tuberculosis strain H37RV.

As part of a study to investigate early changes following exposure to aerosols of Mycobacterium tuberculosis (Mtb), 10 rhesus macaques (Macaca mulatta) were infected with high (731 colony forming units [cfu]), medium (70 cfu) or low (7 cfu) doses of Mtb, and tissues were examined at 2 and 3 weeks post infection (wpi). Clinical disease was not observed. Results of advanced imaging and pathological findings were compared with respect to the delivered dose and time post infection. Magnetic resonance imaging revealed lesions in the lungs at these early time points ex vivo immediately prior to detailed post-mortem examination in the absence of clinical disease. In animals exposed to high and medium doses of Mtb that were studied at 2 and 3 wpi, a range of lesions including small foci of mainly mononuclear cells, primarily macrophages (granulomatous lesions), as well as obvious granulomas, were observed microscopically in the lungs, including lymphatics and hilar lymph nodes. In the low-dose group at 3 weeks, small lesions were identified in the lung and hilar lymph nodes of one animal, and the remaining two animals in this group had lesions in either lung or hilar lymph node. Acid fast bacilli were demonstrated in the lung and lymph nodes in all animals that received high and medium doses, and the lymph nodes of two animals at the low dose. A dose-dependent effect was observed with increasing dose and time post infection. Furthermore, early dissemination of bacilli to the draining, hilar lymph nodes with concomitant granulomatous lesion formation was observed. By contributing to the recognition of early lesion development due to aerosol challenge of Mtb in the rhesus macaque, this study forms a basis for further investigation of early lesions and may inform the design of future vaccine and therapeutic studies involving early time points in this species.

Abdominal fibrosarcoma associated with a retained surgical swab in a dog.

An abdominal fibrosarcoma surrounding a retained surgical swab was identified in a 3-year-old neutered female rottweiler dog presented with chronic inappetence and lethargy. Laparotomy revealed a mass within the omentum, multiple hepatic masses and enlarged mesenteric lymph nodes. The dog was humanely destroyed and submitted for necropsy examination. Microscopically, the omental mass was consistent with a sarcoma surrounding centrally located fibres of foreign material and was infiltrated by epithelioid macrophages containing intracytoplasmic fibre fragments. Sarcoma tissue was also present in mesenteric lymph nodes, liver, spleen and lungs, and some affected lymph nodes contained intralesional epithelioid macrophages with fibre fragments. Immunohistochemical and electron microscopical examinations were consistent with a diagnosis of fibrosarcoma. By fibre analysis and electron microscopy, the intratumoural fibres were identified as cotton fibres with features identical to those obtained from a surgical swab. To our knowledge this is the first description of an abdominal fibrosarcoma associated with a retained surgical swab in a dog. Other examples of foreign body-associated sarcomas in the veterinary literature are vaccine- and implant-induced sarcomas.