The fusion protein of wild-type canine distemper virus is a major determinant of persistent infection.

The wild-type A75/17 canine distemper virus (CDV) strain induces a persistent infection in the central nervous system but infects cell lines very inefficiently. In contrast, the genetically more distant Onderstepoort CDV vaccine strain (OP-CDV) induces extensive syncytia formation. Here, we investigated the roles of wild-type fusion (F(WT)) and attachment (H(WT)) proteins in Vero cells expressing, or not, the canine SLAM receptor by transfection experiments and by studying recombinants viruses expressing different combinations of wild-type and OP-CDV glycoproteins. We show that low fusogenicity is not due to a defect of the envelope proteins to reach the cell surface and that H(WT) determines persistent infection in a receptor-dependent manner, emphasizing the role of SLAM as a potent enhancer of fusogenicity. However, importantly, F(WT) reduced cell-to-cell fusion independently of the cell surface receptor, thus demonstrating that the fusion protein of the neurovirulent A75/17-CDV strain plays a key role in determining persistent infection.

Activities of fosfomycin and rifampin on planktonic and adherent Enterococcus faecalis strains in an experimental foreign-body infection model.

Enterococcal implant-associated infections are difficult to treat because antibiotics generally lack activity against enterococcal biofilms. We investigated fosfomycin, rifampin, and their combinations against planktonic and adherent Enterococcus faecalis (ATCC 19433) in vitro and in a foreign-body infection model. The MIC/MBClog values were 32/>512 μg/ml for fosfomycin, 4/>64 μg/ml for rifampin, 1/2 μg/ml for ampicillin, 2/>256 μg/ml for linezolid, 16/32 μg/ml for gentamicin, 1/>64 μg/ml for vancomycin, and 1/5 μg/ml for daptomycin. In time-kill studies, fosfomycin was bactericidal at 8× and 16× MIC, but regrowth of resistant strains occurred after 24 h. With the exception of gentamicin, no complete inhibition of growth-related heat production was observed with other antimicrobials on early (3 h) or mature (24 h) biofilms. In the animal model, fosfomycin alone or in combination with daptomycin reduced planktonic counts by ≈4 log10 CFU/ml below the levels before treatment. Fosfomycin cleared planktonic bacteria from 74% of cage fluids (i.e., no growth in aspirated fluid) and eradicated biofilm bacteria from 43% of cages (i.e., no growth from removed cages). In combination with gentamicin, fosfomycin cleared 77% and cured 58% of cages; in combination with vancomycin, fosfomycin cleared 33% and cured 18% of cages; in combination with daptomycin, fosfomycin cleared 75% and cured 17% of cages. Rifampin showed no activity on planktonic or adherent E. faecalis, whereas in combination with daptomycin it cured 17% and with fosfomycin it cured 25% of cages. Emergence of fosfomycin resistance was not observed in vivo. In conclusion, fosfomycin showed activity against planktonic and adherent E. faecalis. Its role against enterococcal biofilms should be further investigated, especially in combination with rifampin and/or daptomycin treatment.

Role of Toll-Like Receptor 9 Signaling in Experimental Leishmania braziliensis Infection.

Infection with Leishmania braziliensis causes cutaneous or mucocutaneous leismaniasis in humans. Toll-like receptor 9 (TLR9) expression has been found in granulomas of lesions in L. braziliensis-infected individuals. L. braziliensis inoculation in mice induces very small lesions that are self-healing, whereas deficiency in the TLR adaptor molecule, MyD88, renders mice susceptible to infection. The TLR involved has not been identified, prompting us to investigate if TLR9 triggering by the parasite contributes to the strong resistance to infection observed in L. braziliensis-inoculated mice. The parasites activated wild-type (WT) dendritic cells (DCs) in vitro but not DCs derived from TLR9(-/-) mice. TLR9(-/-) mice inoculated with L. braziliensis exhibited a transient susceptibility characterized by increased lesion size and parasite burden compared to those of WT mice. Surprisingly, elevated levels of gamma interferon (IFN-γ) were measured at the site of infection and in draining lymph node T cells of TLR9(-/-) mice at the peak of susceptibility, suggesting that unlike observations in vitro, the parasite could induce DC activation leading to the development of Th1 cells in the absence of TLR9 expression. Taken together, these data show that TLR9 signaling is important for the early control of lesion development and parasite burden but is dispensable for the differentiation of Th1 cells secreting IFN-γ, and the high levels of this cytokine are not sufficient to control early parasite replication following L. braziliensis infection.

Antifungal activity against planktonic and biofilm Candida albicans in an experimental model of foreign-body infection.

OBJECTIVES: The treatment of Candida implant-associated infections remains challenging. We investigated the antifungal activity against planktonic and biofilm Candida albicans in a foreign-body infection model. METHODS: Teflon cages were subcutaneously implanted in guinea pigs, infected with C. albicans (ATCC 90028). Animals were treated intraperitoneally 12 h after infection for 4 days once daily with saline, fluconazole (16 mg/kg), amphotericin B (2.5 mg/kg), caspofungin (2.5 mg/kg) or anidulafungin (20 mg/kg). Planktonic Candida was quantified, the clearance rate and cure rate determined. RESULTS: In untreated animals, planktonic Candida was cleared from cage fluid in 25% (infected with 4.5 × 10(3) CFU/cage), 8% (infected with 4.8 × 10(4) CFU/cage) and 0% (infected with 6.2 × 10(5) CFU/cage). Candida biofilm persisted on all explanted cages. Compared to untreated controls, caspofungin reduced the number of planktonic C. albicans to 0.22 and 0.0 CFU/ml, respectively, and anidulafungin to 0.11 and 0.13 CFU/ml, respectively. Fluconazole cured 2/12 cages (17%), amphotericin B and anidulafungin 1/12 cages (8%) and caspofungin 3/12 cages (25%). CONCLUSION: Echinocandins showed superior activity against planktonic C. albicans. Caspofungin showed the highest cure rate of C. albicans biofilm. However, no antifungal exceeded 25% cure rate, demonstrating the difficulty of eradicating Candida biofilms from implants.

Use of a human-like low-grade bacteremia model of experimental endocarditis to study the role of Staphylococcus aureus adhesins and platelet aggregation in early endocarditis.

Animal models of infective endocarditis (IE) induced by high-grade bacteremia revealed the pathogenic roles of Staphylococcus aureus surface adhesins and platelet aggregation in the infection process. In humans, however, S. aureus IE possibly occurs through repeated bouts of low-grade bacteremia from a colonized site or intravenous device. Here we used a rat model of IE induced by continuous low-grade bacteremia to explore further the contributions of S. aureus virulence factors to the initiation of IE. Rats with aortic vegetations were inoculated by continuous intravenous infusion (0.0017 ml/min over 10 h) with 10(6) CFU of Lactococcus lactis pIL253 or a recombinant L. lactis strain expressing an individual S. aureus surface protein (ClfA, FnbpA, BCD, or SdrE) conferring a particular adhesive or platelet aggregation property. Vegetation infection was assessed 24 h later. Plasma was collected at 0, 2, and 6 h postinoculation to quantify the expression of tumor necrosis factor (TNF), interleukin 1α (IL-1α), IL-1β, IL-6, and IL-10. The percentage of vegetation infection relative to that with strain pIL253 (11%) increased when binding to fibrinogen was conferred on L. lactis (ClfA strain) (52%; P = 0.007) and increased further with adhesion to fibronectin (FnbpA strain) (75%; P < 0.001). Expression of fibronectin binding alone was not sufficient to induce IE (BCD strain) (10% of infection). Platelet aggregation increased the risk of vegetation infection (SdrE strain) (30%). Conferring adhesion to fibrinogen and fibronectin favored IL-1β and IL-6 production. Our results, with a model of IE induced by low-grade bacteremia, resembling human disease, extend the essential role of fibrinogen binding in the initiation of S. aureus IE. Triggering of platelet aggregation or an inflammatory response may contribute to or promote the development of IE.

NALP3 is not necessary for early protection against experimental tuberculosis.

In vitro, Toll-like receptors (TLR)2, 4 and 9 as well as NOD-like receptor 2 critically determine macrophage responses to Mycobacterium tuberculosis (Mtb) infection. However, in low-dose experimental murine tuberculosis, single or multiple deficiencies in TLRs 2, 4, 9 or NOD2 have little, if any, impact on early mycobacterial growth containment, granuloma formation and survival. Here, we analyzed the relevance of NALP3, one component of the danger-signaling inflammasome, for (i) Mtb-induced cytokine secretion in vitro and in vivo, (ii) restriction of Mtb replication in infected organs and (iii) granuloma formation. In the absence of functional NALP3, there was no IL-1beta and IL-18 production in Mtb-infected dendritic cells and macrophages in vitro, whereas secretion of IL-1alpha, IL-12p40 and TNF remained unaffected. After three weeks of infection, NALP3-deficient as well as IL-18-deficient mice were as capable as wildtype mice of restricting Mtb loads at a plateau level within well-differentiated granulomas. In conclusion, despite its involvement in cytokine processing, NALP3 is not essential for induction of protective immunity to Mtb.

The murine model of infection with Leishmania major and its importance for the deciphering of mechanisms underlying differences in Th cell differentiation in mice from different genetic backgrounds.

Mice from the majority of inbred strains are resistant to infection by Leishmania major, an obligate intracellular protozoan parasite of macrophages in the mammalian host. In contrast, mice from BALB strains are unable to control infection and develop progressive disease. In this model of infection, genetically determined resistance and susceptibility have been clearly shown to result from the appearance of parasite-specific CD4+ T helper 1 or T helper 2 cells, respectively. This murine model of infection is considered as one of the best experimental systems for the study of the mechanisms operating in vivo at the initiation of polarised T helper 1 and T helper 2 cell maturation. Among the several factors influencing Th cell development, cytokines themselves critically regulate this process. The results accumulated during the last years have clarified some aspects of the role played by cytokines in Th cell differentiation. They are providing critical information that may ultimately lead to the rational devise of means by which to tailor immune responses to the effector functions that are most efficient in preventing and/or controlling infections with pathogens.

Agglutinating Secretory IgA Preserves Intestinal Epithelial Cell Integrity during Apical Infection by Shigella flexneri.

Shigella flexneri, by invading intestinal epithelial cells (IECs) and inducing inflammatory responses of the colonic mucosa, causes bacillary dysentery. Although M cells overlying Peyer's patches are commonly considered the primary site of entry of S. flexneri, indirect evidence suggests that bacteria can also use IECs as a portal of entry to the lamina propria. Passive delivery of secretory IgA (SIgA), the major immunoglobulin secreted at mucosal surfaces, has been shown to protect rabbits from experimental shigellosis, but no information exists as to its molecular role in maintaining luminal epithelial integrity. We have established that the interaction of virulent S. flexneri with the apical pole of a model intestinal epithelium consisting of polarized Caco-2 cell monolayers resulted in the progressive disruption of the tight junction network and actin depolymerization, eventually resulting in cell death. The lipopolysaccharide (LPS)-specific agglutinating SIgAC5 monoclonal antibody (MAb), but not monomeric IgAC5 or IgGC20 MAbs of the same specificity, achieved protective functions through combined mechanisms, including limitation of the interaction between S. flexneri and epithelial cells, maintenance of the tight junction seal, preservation of the cell morphology, reduction of NF-κB nuclear translocation, and inhibition of proinflammatory mediator secretion. Our results add to the understanding of the function of SIgA-mediated immune exclusion by identifying a mode of action whereby the formation of immune complexes translates into maintenance of the integrity of epithelial cells lining the mucosa. This novel mechanism of protection mediated by SIgA is important to extend the arsenal of effective strategies to fight against S. flexneri mucosal invasion.

Early DNA vaccination of puppies against canine distemper in the presence of maternally derived immunity.

Canine distemper (CD) is a disease in carnivores caused by CD virus (CDV), a member of the morbillivirus genus. It still is a threat to the carnivore and ferret population. The currently used modified attenuated live vaccines have several drawbacks of which lack of appropriate protection from severe infection is the most outstanding one. In addition, puppies up to the age of 6-8 weeks cannot be immunized efficiently due to the presence of maternal antibodies. In this study, a DNA prime modified live vaccine boost strategy was investigated in puppies in order to determine if vaccinated neonatal dogs induce a neutralizing immune response which is supposed to protect animals from a CDV challenge. Furthermore, a single DNA vaccination of puppies, 14 days after birth and in the presence of high titers of CDV neutralizing maternal antibodies, induced a clear and significant priming effect observed as early as 3 days after the subsequent booster with a conventional CDV vaccine. It was shown that the priming effect develops faster and to higher titers in puppies preimmunized with DNA 14 days after birth than in those vaccinated 28 days after birth. Our results demonstrate that despite the presence of maternal antibodies puppies can be vaccinated using the CDV DNA vaccine, and that this vaccination has a clear priming effect leading to a solid immune response after a booster with a conventional CDV vaccine.

Experimental evaluation of an electromechanical artificial urinary sphincter in an animal model.

WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?: The AMS 800 urinary control system is the gold standard for the treatment of urinary incontinence due to sphincter insufficiency. Despite excellent functional outcome and latest technological improvements, the revision rate remains significant. To overcome the shortcomings of the current device, we developed a modern electromechanical artificial urinary sphincter. The results demonstrated that this new sphincter is effective and well tolerated up to 3 months. This preliminary study represents a first step in the clinical application of novel technologies and an alternative compression mechanism to the urethra. OBJECTIVES: To evaluate the effectiveness in continence achievement of a new electromechanical artificial urinary sphincter (emAUS) in an animal model. To assess urethral response and animal general response to short-term and mid-term activation of the emAUS. MATERIALS AND METHODS: The principle of the emAUS is electromechanical induction of alternating compression of successive segments of the urethra by a series of cuffs activated by artificial muscles. Between February 2009 and May 2010 the emAUS was implanted in 17 sheep divided into three groups. The first phase aimed to measure bladder leak point pressure during the activation of the device. The second and third phases aimed to assess tissue response to the presence of the device after 2-9 weeks and after 3 months respectively. Histopathological and immunohistochemistry evaluation of the urethra was performed. RESULTS: Bladder leak point pressure was measured at levels between 1091 ± 30.6 cmH2 O and 1244.1 ± 99 cmH2 O (mean ± standard deviation) depending on the number of cuffs used. At gross examination, the explanted urethra showed no sign of infection, atrophy or stricture. On microscopic examination no significant difference in structure was found between urethral structure surrounded by a cuff and control urethra. In the peripheral tissues, the implanted material elicited a chronic foreign body reaction. Apart from one case, specimens did not show significant presence of lymphocytes, polymorphonuclear leucocytes, necrosis or cell degeneration. Immunohistochemistry confirmed the absence of macrophages in the samples. CONCLUSIONS: This animal study shows that the emAUS can provide continence. This new electronic controlled sequential alternating compression mechanism can avoid damage to urethral vascularity, at least up to 3 months after implantation. After this positive proof of concept, long-term studies are needed before clinical application could be considered.

Parenteral sparfloxacin compared with ceftriaxone in treatment of experimental endocarditis due to penicillin-susceptible and -resistant streptococci.

A new, investigational, parenteral form of sparfloxacin was compared with ceftriaxone in the treatment of experimental endocarditis caused by either of three penicillin-susceptible streptococci or one penicillin-resistant streptococcus. Both drugs have prolonged half-lives in serum, allowing single daily administration to humans. Sparfloxacin had relatively low MICs (0.25 to 0.5 mg/liter) for all four organisms and was also greater than or equal to eight times more effective than the other quinolones against 21 additional streptococcal isolates recovered from patients with bacteremia. Ceftriaxone MICs were 0.032 to 0.064 mg/liter for the penicillin-susceptible strains and 2 mg/liter for the resistant isolate. Both antibiotics resulted in moderate bacterial killing in vitro. Rats with catheter-induced aortic vegetations were inoculated with 10(7) CFU of the test organisms. Antibiotic treatment was started 48 h later and lasted either 3 or 5 days. The drugs were injected at doses which mimicked the kinetics in human serum produced by one intravenous injection of 400 mg of sparfloxacin (i.e., the daily dose expected to be given to human adults) and 2 g of ceftriaxone. Both antibiotics significantly decreased the bacterial densities in the vegetations. However, sparfloxacin was slower than ceftriaxone in its ability to eradicate valvular infection caused by penicillin-susceptible bacteria. While this difference was quite marked after 3 days of therapy, it tended to vanish when treatment was prolonged to 5 days. In contrast, sparfloxacin was very effective against the penicillin-resistant isolate, an organism against which ceftriaxone therapy failed in vivo. No sparfloxacin-resistant mutant was selected during therapy. Thus, in the present experimental setting, this new, investigational, parenteral form of sparfloxacin was effective against severe infections caused by both penicillin-susceptible and penicillin-resistant streptococci.

The protective capacities of histone H1 against experimental murine cutaneous leishmaniasis.

In a murine model of experimental cutaneous leishmaniasis, we investigated the protection elicited by injection of histone H1 isolated from parasites by perchloric extraction, of a H1 recombinant protein produced in E. coli, and of H1 long and short synthetic peptides, against infection by L. major. Partial protection was achieved in most of the animals as shown by reduction in lesion size, upon immunization with histone H1 or its peptides, provided that the region 1-60 was present in the molecule. These observations argue in favor of a thorough examination of the possibility of including histone H1 described here in a cocktail vaccine against human leishmaniasis.

Contribution of clumping factor B to pathogenesis of experimental endocarditis due to Staphylococcus aureus.

Staphylococcus aureus Newman with an insertion mutation in clfB, the gene encoding clumping factor B, only marginally decreased infection rate (P&gt;0.05) in rats with experimental endocarditis. In contrast, clfB complementation on a multicopy plasmid significantly increased infectivity (P&lt;0.05) over the deleted mutants. Although clfB could affect endovascular infection, its importance in experimental endocarditis was limited.

Efficacies of moxifloxacin, ciprofloxacin, and vancomycin against experimental endocarditis due to methicillin-resistant Staphylococcus aureus expressing various degrees of ciprofloxacin resistance.

The new 8-methoxyquinolone moxifloxacin was tested against two ciprofloxacin-susceptible Staphylococcus aureus strains (strains P8 and COL) and two ciprofloxacin-resistant derivatives of strain P8 carrying a single grlA mutation (strain P8-4) and double grlA and gyrA mutations (strain P8-128). All strains were resistant to methicillin. The MICs of ciprofloxacin and moxifloxacin were 0.5 and 0.125 mg/liter, respectively, for P8; 0.25 and 0.125 mg/liter, respectively, for COL; 8 and 0.25 mg/liter, respectively, for P8-4; and &gt;or=128 and 2 mg/liter, respectively, for P8-128. In vitro, the rate of spontaneous resistance of P8 and COL was 10(-7) on agar plates containing ciprofloxacin at two times the MIC, whereas it was &lt;or=10(-10) on agar plates containing moxifloxacin at two times the MIC. Rats with experimental aortic endocarditis were treated with doses of drugs that simulate the kinetics in humans: moxifloxacin, 400 mg orally once a day; ciprofloxacin, 750 mg orally twice a day; or vancomycin, 1 g intravenously twice a day. Treatment was started either 12 or 24 h after infection and lasted for 3 days. Moxifloxacin treatment resulted in culture-negative vegetations in a total of 20 of 21 (95%) rats infected with P8, 10 of 11 (91%) rats infected with COL, and 19 of 24 (79%) rats infected with P8-4 (P &lt; 0.05 compared to the results for the controls). In contrast, ciprofloxacin treatment sterilized zero of nine (0%) vegetations infected with first-level resistant mutant P8-4. Vancomycin sterilized only 8 of 15 (53%), 6 of 11 (54%), and 12 of 23 (52%) of the vegetations, respectively. No moxifloxacin-resistant derivative emerged among these organisms. However, moxifloxacin treatment of highly ciprofloxacin-resistant mutant P8-128 failed and selected for variants for which the MIC increased two times in 2 of 10 animals. Thus, while oral moxifloxacin might deserve consideration as treatment for staphylococcal infections in humans, caution related to its use against strains for which MICs are borderline is warranted.

RP 59500 prophylaxis of experimental endocarditis due to erythromycin-susceptible and -resistant isogenic pairs of viridans group streptococci.

RP 59500 is a new injectable streptogramin composed of two synergistic components (quinupristin and dalfopristin) which are active against a number of erythromycin-susceptible and -resistant gram-positive bacteria. The following experiments investigate the ability of RP 59500 to prevent experimental endocarditis due to either of two erythromycin-susceptible streptococcal isolates or their constitutively erythromycin-resistant Tn916 delta E transconjugants. RP 59500 had low MICs (0.125 to 0.5 mg/liter) for all four test organisms and was substantially bactericidal in vitro. Rats with catheter-induced aortic vegetations were given single-dose antibiotic prophylaxis 30 to 60 min before bacterial inoculation through a computerized pump system which permitted the simulation of drug kinetics for humans produced by either 7 mg of RP 59500 per kg of body weight or 1 g of vancomycin. Single-dose RP 59500 prophylaxis successfully prevented endocarditis due to both the erythromycin-susceptible parent strains and their erythromycin-resistant derivatives in rats challenged with the minimal inoculum infecting 90% of controls. In addition, RP 59500 also prevented infection in animals challenged with fivefold-larger inocula of the erythromycin-susceptible parent strains. Vancomycin successfully prevented endocarditis due to any of the four test organisms. These results underline the in vivo efficacy of RP 59500 against both erythromycin-susceptible and -resistant streptococci. Such good results against the resistant strains would not be expected with erythromycin or clindamycin, which are the standard macrolidelincosamide-streptogramin antibiotics used for endocarditis prophylaxis in humans. An oral form of RP 59500 which might advantageously replace some of the older prophylactic regimens is currently being developed.