Aerosol therapy of influenza infections of mice and primates with rimantadine, ribavirin, and related compounds.

Ribavirin administered as small-article aerosols had significant therapeutic effect in the treatment of viral respiratory infections induced by influenza virus. The preliminary experiment using ribavirin to treat influenza infection in the squirrel monkey is encouraging. We expect to extend these experiments by initiating therapy at a later time to investigate the potential value of ribavirin in a clinical situation. Several derivatives of ribavirin are effective antiviral compounds. The tri-O-acetyl derivative appears to offer a potential advantage over ribavirin, although this cannot be stated with certainty since the data were obtained from separate experiments. Radiolabeling has been used as a means of measuring tissue concentration and clearance rates of various drugs. It is hoped that the use of labeled ribavirin and the tri-O-acetyl derivative will assist us in determining whether a depot of antiviral drug is created in pulmonary tissues after administration as a small-particle aerosol. These experiments are now in progress.

Treatment of Klebsiella pneumoniae respiratory tract infection of squirrel monkeys with aerosol administration of kanamycin.

The therapeutic efficacy of IM-administered kanamycin was compared with the efficacy of aerosol-administered kanamycin in Klebsiella pneumoniae-infected squirrel monkeys. Differences in mortality or morbidity were not seen with equivalent dosages of antibiotic ranging from 15 to 6.9 mg/kg of body weight/day. Seemingly, the IM route of kanamycin administration was as effective as the aerosol route for therapy.

Protection against Klebsiella pneumoniae respiratory tract infection of mice and squirrel monkeys given kanamycin by aerosol and injection.

The methods of aerosol administration of kanamycin and IM injection of the antibiotic were compared for their protection of mice and squirrel monkeys against Klebsiella pneumoniae respiratory tract infection. Mice exposed to LD90 of K pneumoniae at 0.5, 4, 24, 48 and 72 hours after they were treated with aerosol of kanamycin (27 mg/kg of body weight) were significantly better protected at all exposure times than were mice given the antibiotic (450 mg/kg) by IM injection. Squirrel monkeys given the aerosol at dose level of 11.25 mg/kg were completely protected against K pneumoniae exposure at 6 and 24 hours, whereas only one of eight monkeys treated with the same dose given IM survived the exposure at 6 hours and none survived at 24 hours. Antibiotic clearance curves indicated that kanamycin remained in the lungs at higher concentrations and for longer periods after aerosol treatment than after IM treatment.

Influence of blue-green algae (cyanobacteria) on survival of Legionella pneumophila in aerosols.

The fluid in which blue-green algae (cyanobacteria) (Fischerella sp.) had been grown (algal extract) was investigated for its effect on aerosols of Legionella pneumophila. The bacteria were significantly more stable when suspended in algal extract than in the tryptose-saline solution employed in previously reported experiments. The stabilizing property of the extract disappeared after dialysis, suggesting that a relatively small molecule was involved. The relationship of this observation to the epidemiology of Legionnaires disease is discussed.

Relationship of method of administration to respiratory virulence of Klebsiella pneumoniae for mice and squirrel monkeys.

Klebsiella pneumoniae given by aerosol was significantly less virulent in mice and monkeys than when given by intranasal (mice) or intratracheal (monkeys) instillation.

Simian model for the evaluation of immunity to influenza.

Rhesus monkeys (Macaca mulatta) exposed to small-particle aerosols of the Aichi strain of type A2 influenza virus responded by shedding virus from the nasopharynx for 7 to 9 days and by seroconversion (hemagglutination inhibition) 8 or 9 days after exposure. After rechallenge with the homologous virus, no replication of the organism was observed, and a serological anamnestic reaction occurred. The data indicate that the rhesus monkey is a useful primate model for evaluating induced immunity to influenza virus infection.

Effect of simulated solar radiation and sodium fluorescein on the recovery of Venezuelan equine encephalomyelitis virus from aerosols.

Almost 90% of the Trinidad strain of Venezuelan equine encephalomyelitis (VEE) virus survived for 1 hr after aerosolization into a dark environment at 30% relative humidity (RH), and 78% survived for 1 hr at 60% RH. After exposure to simulated solar radiation (584 mcal per cm(2) per min) 0.02% of the aerosolized virus survived for 1 hr at 30% RH and 0.006% survived for 1 hr at 60% RH. When 1.0 mg of sodium fluorescein per ml was added to suspensions prior to aerosol dissemination (to determine physical loss of aerosol), no virus was detected after 30 min at either RH upon irradiation. Sodium fluorescein also exhibited some toxicity (31% survival at 60 min) for nonirradiated aerosols of VEE virus at 60% RH; no effect was noted at 30%.

Experimental tularemia in Macaca mulatta: relationship of aerosol particle size to the infectivity of airborne Pasteurella tularensis.

Ninety-six Macaca mulatta were exposed to aerosol particles containing Pasteurella tularensis. Four different aerosols were employed that contained particle size distributions with median diameters of 2.1, 7.5, 12.5, or 24.0 mum. Size distributions were calculated only for those particles observed by phase microscope to contain organisms. Animals exposed to particles whose median diameters were either 2.1 or 7.5 mum were all infected and showed extensive infection of the lower respiratory tract, evidenced by large patches of consolidation with many necrotic foci on the surface. Death occurred in these animals 4 to 8 days after exposure. Monkeys exposed to 12.5- or 24.0-mum median diameter particles presented involvement of the cervical and mandibular lymph nodes, evidenced by swelling and abscess formation. Thirty-eight of the 45 animals in this group were infected. Those animals succumbing to the disease died from 8 to 21 days after exposure. The respiratory LD(50) values increased from 14 to 4,447 cells as the median diameter was raised from 2.1 to 24.0 mum.

Evaluation of commercially prepared vaccines for experimentally induced type A/New Jersey/8/76 influenza virus infections in mice and squirrel monkeys.

Mice and squirrel monkeys were vaccinated and subsequently challenged at selected times to evaluate the immunoprophylactic value of vaccines against influenza virus type A/New Jersey/76. Mice were challenged with virulent, homologous virus either 17 or 60 days after vaccination with 80 chick cell-agglutinating (CCA) units of whole-virus vaccine. Vaccinated mice showed minimal lesions and virus in lung tissue and had lower lung weights than unvaccinated controls. These mice had titers of hemagglutination-inhibiting (HAI) antibody in serum of greater than 1:400, but only traces of antibody were found in lung washes. Vaccinated squirrel monkeys had significantly less illness than unvaccinated controls when challenged with virulent virus 30 days after intramuscular immunization with 200 CCA units of whole virus or 400 CCA units of split virus given either once or twice (at 30-day intervals). Equal protection was observed in all monkeys despite the absence of serum HAI antibody in some monkeys after vaccination. Anamnestic reactions were observed only in monkeys vaccinated with whole virus. The possible roles of various immune factors and antibody to neuraminidase are discussed.

Reaction of squirrel monkeys to intratracheal inoculation with influenza/A/New Jersey/76 (swine) virus.

To determine whether a model could be established for laboratory investigations, nine squirrel monkeys were inoculated intratracheally with 10(7) median egg-infectious doses of influenza virus type A/New Jersey/8/76 (HSW1N1) (swine influenza virus). They responded with clinically detectable illness including fever, leukopenia, decreased food consumption, increased respiratory rate, occasional coughing, labored breathing, nasal discharge, and lethargy. Convalescence was well advanced by the day 10. All monkeys excreted virus for 7 to 8 days. A scoring procedure (illness score) has been developed for use in studies of vaccine and chemotherapeutic efficacy.

Effect of prior influenza virus infections on susceptibility of AKR/J mice to respiratory challenge with Legionella pneumophila.

Influenza virus administered intranasally to AKR/J mice, followed 3 days later by Legionella pneumophila inoculated intranasally, caused significantly greater mortality than did either of the two agents administered alone. Viable concentrations of both bacteria and viruses dropped in sequentially infected animals, despite the ultimate fatal outcome. Viral concentrations, however, did not decrease as rapidly in sequentially infected as in singly infected mice. Histopathologic lesions were consistent with viral replication aided by elaboration of a bacterial toxin. This observation contrasts with the more commonly observed sequence in which the bacterium proliferates after the virus interferes with host defense. Cell-free preparations were found to have toxic activity.

Nonhuman primate model for the study of respiratory Klebsiella pneumoniae infection.

Squirrel monkeys were inoculated by the intratracheal inoculation of 700 Klebsiella pneumoniae organisms and developed lobar pneumonia in about 24 h. Characteristic clinical findings were fever, anorexia, and coughing. Laboratory findings included leukocytosis or leukopenia (with the latter more prominent in ultimately fatal infections), bacteremia, and shedding of bacteria into the pharynx. Infected monkeys showed increased plasma lysozyme activity as well as increased plasma ceruloplasmin, haptoglobin and alpha1-antitrypsin. The mortality rate was 60%, and the mean time of death was 50.5 h. Pathologically, the disease spread by means of Kohn's pores and other pathways that generally did not involve airways as a means of dissemination until about 30 h. Squirrel monkeys seem to be better models for human respiratory K. pneumoniae infection than rats or mice.

Activity of amantadine, rimantadine, and ribavirin against swine influenza in mice and squirrel monkeys.

Amantadine, rimantadine, and ribavirin given orally, either prophylactically or therapeutically, reduced mortality and increased the survival time of 3-week-old mice infected with the type A/New Jersey/8/76 (swine) strain of influenza virus. In addition, amantadine and rimantadine, administered therapeutically, increased the rate of virus clearance from lungs of infected mice. Administration of amantadine either before or after virus challenge ameliorated the illness in squirrel monkeys; when administered therapeutically, it appeared to eliminate virus shedding from infected monkeys within hours after therapy was initiated.

Treatment of respiratory Klebsiella pneumoniae infection in mice with aerosols of kanamycin.

Aerosols of kanamycin resulted in greater survival of mice challenged with respiratory Klebsiella pneumoniae than the same dosage given intramuscularly. Determinations of viable bacteria in the blood and lungs revealed that aerosolized kanamycin was most effective when infection was confined to the lungs. After the organisms had spread to other areas, however, aerosol therapy was still more effective than intramuscular therapy, but only one-half the infected mice survived.

Influenza alone and in sequence with pneumonia due to Streptococcus pneumoniae in the squirrel monkey.

Squirrel monkeys (Saimiri sciureus) inoculated intratracheally with 10(4.2)-10(8.2) egg median infectious doses (EID50) of type A influenza virus (H3N2) responded with clinical illness including such signs as fever, sneezing or coughing, coryza, and increased respiratory rates. Necropsy studies performed six days after inoculation revealed bronchopneumonia in addition to a mild tracheitis. Squirrel monkeys given 10(5)-6 x 10(8) colony-forming units (cfu) of Streptococcus pneumoniae intratracheally died four to six days later after developing severe illness characterized by fever, bacteremia, lethargy, anorexia, coughing, labored breathing, and bronchopneumonia. Monkeys given 770 cfu of S. pneumoniae responded with less severe symptoms and survived. Four squirrel monkeys inoculated with 10(8.2) EID50 of virus and then 102 hr later with 770 cfu of S. pneumoniae developed severe disease; three of the four animals died within 40 hr. At necropsy these monkeys had more extensive and severe bronchopneumonia than was seen in monkeys infected with either organism alone.

Effect of sodium fluorescein and plating medium on recovery of irradiated Escherichia coli and Serratia marcescens from aerosols.

Irradiation of aerosols of either Escherichia coli or Serratia marcescens with simulated solar (xenon) radiation caused a significant decrease in viability. When sodium fluorescein was employed to determine the physical loss of organisms from the aerosol, an additional adverse effect upon survival was noted. The decay curves indicated that at least two mechanisms of inactivation were operative, one due to aerosolization, the other to irradiation. After collection from aerosols, both species of microorganisms grew better on blood agar base than on Casitone agar, but this finding did not appear to be related to the effect of irradiation.

Persistence of Diplococcus pneumoniae after influenza virus infection in Macaca mulatta.

Exposure of monkeys to aerosols of influenza virus followed later by aerosols of Diplococcus pneumoniae resulted in persistence of the bacteria in the upper respiratory tract for an average of 28 days. Examination of selected tissues for pneumococci revealed that pneumococci were widely disseminated throughout the respiratory tract at 3 and 7 days after exposure, but were cleared from lower respiratory tissues and were found mainly in tonsils, oropharynx, and posterior nasopharynx after 14 days.

Dose-response of guinea pigs experimentally infected with aerosols of Legionella pneumophila.

Experiments were carried out to determine the infectivity, clinical course of disease, and lethality of aerosols of Legionella pneumophila for guinea pigs. The median infectious dose was less than 129 organisms; the 50% lethal dose was 1.4 x 10(5) organisms. In addition, the intraperitoneal 50% lethal dose was 3.0 x 10(6) cells, a value indicating that the organisms were less virulent by the intraperitoneal route than by aerosol. Nonfatal disease always included fever and weight loss. These signs were accompanied by sporadic bacteremia and dyspnea. Leukocyte counts were uninformative. In general, the severity of fever and extent of serologic (microagglutination titer) response were dose-related. The guinea pig may be used as a model for Legionnaires' disease, but the only dependable clinical criteria of infection after airborne challenge are weight loss, fever, and seroconversion.

Disseminated intravascular coagulopathy during experimental pneumococcal sepsis: studies in normal and asplenic rhesus monkeys.

Disseminated intravascular coagulation (DIC) was induced in both normal and asplenic rhesus monkeys by intravenous challenge with Streptococcus pneumoniae. Our observations in the infected monkeys have led us to conclude that (1) pneumococcal capsular polysaccharide (PCP), immune complexes and complement may not have primary roles in the initiation of DIC; (2) intact pneumococci may be catalysts for the development of DIC; (3) the initial event in DIC may be activation of Hageman factor; and (4) evidence of activation of Hageman factor-dependent systems is present regardless of severity of infection.

Response of influenza virus-infected mice to selected doses of ribavirin administered intraperitoneally or by aerosol.

The effects of graded doses of ribavirin administered either by aerosol or intraperitoneally were compared in influenza virus-infected mice. The median effective dose values (based upon percent survival) were 3.3 and 15.8 mg/kg per day for the aerosol and intraperitoneal routes, respectively. Lung lesion scores and titer of virus were lower after aerosol than intraperitoneal therapy.