Evaluation of Effects of Laboratory Disinfectants on Mouse Gut Microbiota.

Disturbances in the gut microbiota are known to be associated with numerous human diseases. Mice have proven to be an invaluable tool for investigating the role of the gut microbiota in disease processes. Nonexperimental factors related to maintaining mice in the laboratory environment are increasingly being shown to have inadvertent effects on the gut microbiota and may function as confounding variables. Microisolation technique is a term used to describe the common biosecurity practice of spraying gloved hands with disinfectant before handling research mice. This practice prevents contamination with pathogenic microorganisms. To investigate if exposure to disinfectants can affect the mouse gut microbiota, C57BL/6 mice were exposed daily for 27 consecutive days to commonly used laboratory disinfectants through microisolation technique. The effects of 70% ethanol and disinfectant products containing chlorine dioxide, hydrogen peroxide, or potassium peroxymonosulfate were each evaluated. Fecal pellets were collected after 7, 14, 21, and 28 d of disinfectant exposure, and cecal contents were collected at day 28. DNA extractions were performed on all cecal and fecal samples, and microbial community structure was characterized using 16S ribosomal RNA amplicon sequencing. Alpha and ß diversity metrics and taxon-level analyses were used to evaluate differences in microbial communities. Disinfectant had a small but significant effect on fecal microbial communities compared with sham-exposed controls, and effects varied by disinfectant type. In general, longer exposure times resulted in greater changes in the fecal microbiota. Effects on the cecal microbiota were less pronounced and only seen with the hydrogen peroxide and potassium peroxymonosulfate disinfectants. These results indicate that laboratory disinfectant use should be considered as a potential factor that can affect the mouse gut microbiota.

Effects of Intracage Ammonia on Markers of Pulmonary Endothelial Integrity in Mice Housed in Static Microisolation Cages.

Time-weighted exposure limits to ammonia are established for humans; however similar guidelines have not been defined for laboratory rodents. The Guide recommends maintaining air pollutants at concentrations below levels irritating to mucous membranes but does not provide specific values. Numerous studies have examined ammonia and its effects on animal health, yet none have assessed the effects of naturally occurring intracage ammonia on the lower pulmonary tree and pulmonary endothelial and epithelial integrity in mice. We performed several assays commonly used in mouse acute lung-injury studies (bronchoalveolar lavage fluid [BAL] cell counts and protein concentration, excess lung water content [ELW], Evans blue permeability assay [EBA], lung tissue myeloperoxidase assay [MPO], and lung histopathology) to evaluate the effects of exposure to cyclical, naturally occurring ammonia levels on pulmonary integrity and inflammation. C57BL/6 mice were maintained in static microisolation or open-top cages. Cages were changed weekly, and ammonia levels were measured for 6 wk on days 0, 1, 3, 5, and 7 of each cage-change cycle. Ammonia levels in static microisolation cages began to increase on day 3 and peaked at a mean of 141.3 ppm on day 7. Ammonia levels in open-top cages never exceeded 5 ppm. Neither BAL cell counts, protein concentration, ELW, EBA, nor MPO differed significantly between groups. Lung histopathology showed minimal, incidental changes in all mice. Our findings indicate that the ammonia concentrations in the static microisolation cages we used did not alter the integrity of the lower pulmonary tract nor influence key indicators used to assess acute lung injury.

Cross-Foster Rederivation Compared with Antibiotic Administration in the Drinking Water to Eradicate Bordetella pseudohinzii.

Bordetella pseudohinzii is a microbial agent of potential importance in mice and has confounded pulmonary research at our institution. The purpose of this study was to evaluate cross-foster rederivation and antibiotic administration in the drinking water as methods to eradicate B. pseudohinzii. To evaluate the efficacy of cross-foster rederivation, 29 litters representing 16 strains of mice were cross-fostered from cages positive for B. pseudohinzii to B. pseudohinzii-negative Crl:CD1-Elite surrogate dams. To evaluate antibiotic administration, sulfamethoxazole and trimethoprim (TMS; 0.66 and 0.13 mg/mL, respectively) and tetracycline (4.5 mg/mL) were administered in the drinking water. We assessed 3 antibiotic treatment groups with 12 B. pseudohinzii-positive cages per group (6 cages of CD1 and 6 cages of C57BL/6 mice): TMS for 4 wk, TMS for 6 wk, and tetracycline for 6 wk. Of the 29 litters that underwent cross-foster rederivation, 24 were negative for B. pseudohinzii. Five of the 12 cages treated with TMS for 4 wk and 1 of the 12 cages treated with TMS for 6 wk were negative for B. pseudohinzii at 2 wk after treatment. Three of the 12 cages treated with tetracycline were negative for B. pseudohinzii at 2 wk after treatment. Pearson χ2 analysis revealed significant association between the method of eradication (cross-foster rederivation compared with antibiotic administration) and B. pseudohinzii infection, and an odds-ratio estimate from a logistic regression demonstrated that cross-foster rederivation was more successful. Whereas antibiotic administration in the drinking water failed to eradicate B. pseudohinzii, cross-foster rederivation was successful and has been used to establish a B. pseudohinzii-negative barrier.

Bordetella pseudohinzii as a Confounding Organism in Murine Models of Pulmonary Disease.

A group studying acute lung injury observed an increased percentage of neutrophils in the bronchoalveolar lavage (BAL) fluid of mice. BAL was performed, and lung samples were collected sterilely from 5 C57BL/6 mice that had been bred inhouse. Pure colonies of bacteria, initially identified as Bordetella hinzii were cultured from 2 of the 5 mice which had the highest percentages of neutrophils (21% and 26%) in the BAL fluid. Subsequent sequencing of a portion of the ompA gene from this isolate demonstrated 100% homology with the published B. pseudohinzii sequence. We then selected 10 mice from the investigator's colony to determine the best test to screen for B. pseudohinzii in the facility. BAL was performed, the left lung lobe was collected for culture and PCR analysis, the right lung lobe and nasal passages were collected for histopathology, an oral swab was collected for culture, and an oral swab and fecal pellets were collected for PCR analysis. B. pseudohinzii was cultured from the oral cavity, lung, or both in 8 of the 10 mice analyzed. All 8 of these mice were fecal PCR positive for B. pseudohinzii; 7 had increased neutrophils (5% to 20%) in the BAL fluid, whereas the 8th mouse had a normal neutrophil percentage (2%). Active bronchopneumonia was not observed, but some infected mice had mild to moderate rhinitis. B. pseudohinzii appears to be a microbial agent of importance in mouse colonies that can confound pulmonary research. Commercial vendors and institutions should consider colony screening, routine reporting, and exclusion of B. pseudohinzii.

Pseudomonas aeruginosa induced lung injury model.

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Correction: Persistence of Borrelia burgdorferi in Rhesus Macaques following Antibiotic Treatment of Disseminated Infection.

[This corrects the article on p. e29914 in vol. 7.].

Persistence of Borrelia burgdorferi in rhesus macaques following antibiotic treatment of disseminated infection.

The persistence of symptoms in Lyme disease patients following antibiotic therapy, and their causes, continue to be a matter of intense controversy. The studies presented here explore antibiotic efficacy using nonhuman primates. Rhesus macaques were infected with B. burgdorferi and a portion received aggressive antibiotic therapy 4-6 months later. Multiple methods were utilized for detection of residual organisms, including the feeding of lab-reared ticks on monkeys (xenodiagnosis), culture, immunofluorescence and PCR. Antibody responses to the B. burgdorferi-specific C6 diagnostic peptide were measured longitudinally and declined in all treated animals. B. burgdorferi antigen, DNA and RNA were detected in the tissues of treated animals. Finally, small numbers of intact spirochetes were recovered by xenodiagnosis from treated monkeys. These results demonstrate that B. burgdorferi can withstand antibiotic treatment, administered post-dissemination, in a primate host. Though B. burgdorferi is not known to possess resistance mechanisms and is susceptible to the standard antibiotics (doxycycline, ceftriaxone) in vitro, it appears to become tolerant post-dissemination in the primate host. This finding raises important questions about the pathogenicity of antibiotic-tolerant persisters and whether or not they can contribute to symptoms post-treatment.

Effects of weekly blood collection in C57BL/6 mice.

We assessed hematologic recovery, body weight, and behavior after serial blood collection in 10- to 14-wk-old C57BL/6 mice. Male and female mice (5 to 11 mice for pilot groups, 23 to 35 mice for full study groups) had either 15%, 20%, or 25% of their estimated total blood volume (TBV) collected once weekly for 6 wk. Except for those of the 25% TBV male pilot group, the weights of all mice recovered or increased from one collection to the next. The behavior of all mice, with the exception of the 25% TBV male pilot group, appeared normal throughout the study. Erythrogram value changes from baseline were analyzed at each weekly blood collection. Recovery was defined as the return of mean hemoglobin values to within 2 SD of mean baseline values. According to this definition, mice in the 15% TBV male group and 15%, 20%, and 25% TBV female groups recovered hematologically. To support the statistical definition of recovery, we compared our data with human anemia categories to assess the clinical relevance of the mouse hemoglobin values. On the basis of these data, we conclude that as much as 25% TBV can be collected once weekly from female mice for 6 wk and as much as 15% TBV can be collected once weekly from male mice for 6 wk without producing weight loss, behavioral changes, or clinically significant anemia.

The use of cross-foster rederivation to eliminate murine norovirus, Helicobacter spp., and murine hepatitis virus from a mouse colony.

Over 10 mo, 287 mouse litters were cross-fostered by using 1 of 2 paradigms to eliminate murine norovirus (MNV), Helicobacter spp., murine hepatitis virus (MHV), and Syphacia obvelata. Paradigm 1 involved cross-fostering litters at younger than 48 h with no attention to the changing of bedding material. Paradigm 2 involved cross-fostering litters at younger than 24 h from cages in which the bedding material was changed within 24 h before cross-fostering. After cross-foster rederivation, mice were tested for the presence of Helicobacter spp. by means of fecal PCR at 4, 8, and 12 wk. Surrogates also were tested for MNV by use of multiplex fluorometric assay serology at 4 wk and fecal PCR at 12 wk. Surrogate mice were tested for MHV by means of MFIA at 4 wk and for pinworms by perianal tape test and fecal flotation at 4 and 12 wk. Compared with those from paradigm 1, litters from paradigm 2 were less likely to be positive for MHV and Helicobacter spp. The use of cross-foster rederivation alone was unsuccessful for the elimination of Syphacia obvelata. For cross-foster rederivation, we recommend that litters be younger than 24 h and from cages in which the bedding material was changed within 24 h before cross-fostering. The presence of MNV, Helicobacter spp., and MHV can be predicted reliably at 12, 8, and 4 wk, respectively.

Antigenicity and recombination of VlsE, the antigenic variation protein of Borrelia burgdorferi, in rabbits, a host putatively resistant to long-term infection with this spirochete.

Borrelia burgdorferi, the Lyme disease pathogen, employs several immune-evasive strategies to survive in mammals. Unlike mice, major reservoir hosts for B. burgdorferi, rabbits are considered to be nonpermissive hosts for persistent infection. Antigenic variation of the VlsE molecule is a probable evasion strategy known to function in mice. The invariable region 6 (IR6) and carboxyl-terminal domain (Ct) of VlsE elicit dominant antibody responses that are not protective, perhaps to function as decoy epitopes that protect the spirochete. We sought to determine if either of these characteristics of VlsE differed in rabbit infection, contributing to its reputed nonpermissiveness. VlsE recombination was observed in rabbits that were given inoculations with either cultured or host-adapted spirochetes. Early observations showed a lack of anti-C6 (a peptide encompassing the IR6 region) response in most rabbits, so the anti-Ct and anti-C6 responses were monitored for 98 weeks. Anti-C6 antibody appeared as late as 20 weeks postinoculation, and the anti-Ct response, evident within the first 2 weeks, oscillated for prolonged periods of time. These observations, together with the recovery of cultivable spirochetes from tissue of one animal at 98 weeks postinoculation, challenge the notion that the rabbit cannot harbour a long-term B. burgdorferi infection.

Chronic binge ethanol consumption accelerates progression of simian immunodeficiency virus disease.

BACKGROUND: While alcohol consumption is known to increase the incidence and severity of infections, the impact of alcohol consumption on human immunodeficiency virus (HIV) disease progression has been difficult to assess. Therefore, we examined the effect of ethanol on simian immunodeficiency virus (SIV) disease progression in a well-defined model utilizing rhesus macaques. METHODS: Alcohol was administered for 5 hours via an indwelling intragastric catheter to achieve an alcohol concentration of 50 to 60 mM for 4 consecutive days per week for the duration of the study. Control animals received isocaloric sucrose. After 3 months, animals were inoculated intravenously with 10,000 times the ID(50) of SIV(DeltaB670) and followed to end-stage disease. RESULTS: Plasma SIV ribonucleic acid (RNA) was higher in alcohol-consuming animals compared with sucrose-treated animals during the early asymptomatic stage of disease but not at later time points. This increase in viral set point was associated with more rapid progression to end-stage disease in macaques administered alcohol (median=374 days) compared with sucrose (median=900 days). The decline in blood CD4+ cells was similar in both groups of animals. CONCLUSIONS: This study indicates that frequent episodes of alcohol intoxication in SIV+ macaques increase viral set point in association with more rapid development of end-stage disease.

Experimental infection of rhesus macaques with Streptococcus pneumoniae: a possible model for vaccine assessment.

BACKGROUND: We explored the possibility of using normal adult rhesus macaques for the preclinical assessment of safety, immunogenicity, and efficacy of newly developed vaccines against Streptococcus pneumoniae infection of the lung. METHODS: Our primary objective was to determine whether an intra-bronchial inoculum of at least 10(6)S. pneumoniae colony-forming units, or one as high as 10(8)-10(9) organisms, could detectably survive in rhesus macaques for a period longer than 1-2 weeks. If so, we hypothesized, it would be possible to observe signs of pneumonia commonly observed in humans, and discriminate between vaccinated/protected animals and controls. Infection was detectable in bronchoalveolar lavage fluids 3-5 weeks post-inoculation. RESULTS: The clinical course of disease mimicked aspects of that of human pneumococcal pneumonia. Signs of inflammation typical of the disease in humans, such as elevated concentrations of neutrophils and of pro-inflammatory cytokines in bronchoalveolar lavage fluids were also observed. CONCLUSIONS: These findings underscore the utility of this model to assess the safety, immunogenicity, and efficacy of newly developed S. pneumoniae vaccines.

Induction of Plasmodium falciparum transmission-blocking antibodies in nonhuman primates by a combination of DNA and protein immunizations.

Malaria transmission-blocking vaccination can effectively reduce and/or eliminate transmission of parasites from the human host to the mosquito vector. The immunity achieved by inducing an antibody response to surface antigens of male and female gametes and parasite stages in the mosquito. Our laboratory has developed DNA vaccine constructs, based on Pfs25 (a Plasmodium falciparum surface protein of 25 kDa), that induce a transmission-blocking immune response in mice (C. A. Lobo, R. Dhar, and N. Kumar, Infect. Immun. 67:1688-1693, 1999). To evaluate the safety, immunogenicity, and efficacy of the Pfs25 DNA vaccine in nonhuman primates, we immunized rhesus macaques (Macaca mulatta) with a DNA vaccine plasmid encoding Pfs25 or a Pfg27-Pfs25 hybrid or with the plasmid (empty plasmid) alone. Immunization with four doses of these DNA vaccine constructs elicited antibody titers that were high but nonetheless unable to reduce the parasite's infectivity in membrane feeding assays. Further boosting of the antibody response with recombinant Pfs25 formulated in Montanide ISA-720 increased antibody titers (30-fold) and significantly blocked transmission of P. falciparum gametocytes to Anopheles mosquitoes (approximately 90% reduction in oocyst numbers in the midgut). Our data show that a DNA prime-protein boost regimen holds promise for achieving transmission-blocking immunity in areas where malaria is endemic and could be effective in eradicating malaria in isolated areas where the level of malaria endemicity is low.

Strain-dependent response to cerebral ischemic preconditioning: differences between spontaneously hypertensive and stroke prone spontaneously hypertensive rats.

Ischemic preconditioning (PC) is a phenomenon whereby a brief exposure to ischemia renders a tissue more tolerant to a subsequent sustained ischemic insult. Animals of the Spontaneously Hypertensive (SHR) and the Spontaneously Hypertensive Stroke-Prone (SHR-SP) rat strains produce cerebral infarcts that are larger and more reproducible in size than infarcts of normotensive rats. This study compared the effects of PC in SHR and SHR-SP rats, under the hypothesis that PC may not be as effective in the SHR-SP, a strain genetically predisposed to stroke. There were two groups per strain, with between eight and ten animals each. The Precondition group (PC) had a 10 min occlusion of the middle cerebral artery on day -1. On the same day the Sham group (Sham) received sham surgery. On day 0, both groups underwent permanent occlusion of the middle cerebral artery. The ischemic lesion was measured on day 1 using T(2)-weighted magnetic resonance imaging. Percent hemispheric infarct was significantly reduced in SHR PC vs. SHR Sham, SHR-SP PC vs. SHR-SP Sham, and SHR PC vs. SHR-SP PC. Thus, rats of the SHR-SP strain respond to PC less markedly than SHR animals. Both models may now be used to elucidate the mechanisms underlying PC.

Ixodes scapularis ticks (Acari: Ixodidae) from Louisiana are competent to transmit Borrelia burgdorferi, the agent of Lyme borreliosis.

The principal vector of Borrelia burgdorferi, the Lyme borreliosis spirochete, in the Northeast and Midwestern regions of the United States is the blacklegged tick Ixodes scapularis. Because of a favorable environment, I. scapularis is also plentiful in the South; however, a correlation with Lyme borreliosis cases does not exist in this region of the United States. Concern existed that something intrinsic to ticks found in Louisiana could mitigate their ability to transmit B. burgdorferi. Therefore, we set out to assess the ability of I. scapularis ticks from Louisiana to become infected with and transmit B. burgdorferi using mice as hosts. In the laboratory, mating adult female ticks collected in southeastern Louisiana were fed on the ears of rabbits. After oviposition and egg hatching, the resulting larvae were fed on mice that had been needle-inoculated with two different strains of B. burgdorferi sensu stricto, B31 and JD1. Larvae were found to be positive for spirochetes. Additional fed larvae were allowed to molt into the nymphal stage. Flat nymphs remained infected with B. burgdorferi. Infected nymphs were allowed to feed on naïve mice, all of which became infected as shown by culture of ear biopsy specimens. Naïve larvae were then fed on these same mice to assess transmissibility. The resulting engorged larvae harbored spirochetes. We have demonstrated that the I. scapularis ticks found in Louisiana are fully competent to carry and transmit B. burgdorferi infection.

Alcohol unmasks simian immunodeficiency virus-induced cognitive impairments in rhesus monkeys.

BACKGROUND: Alcohol and human immunodeficiency virus (HIV) produce similar neuropathological profiles, including loss of neurons in the frontal cortex. Additionally, HIV-positive patients with a history of alcohol abuse have greater neurologic deficits, and chronic alcohol abuse produces electrophysiological deficits earlier in the HIV disease process. Few studies, preclinical or clinical, have examined whether alcohol administration exacerbates the neuropsychological deficits in subjects with lentiviruses such as HIV. METHODS: To examine the combined effects of alcohol and immunodeficiency viruses (IVs) on neuropsychological functioning, four groups of young adult rhesus monkeys trained to respond under two complex behavioral tasks were administered ethanol 4 days per week via an intragastric catheter for 3 months and then infected with simian immunodeficiency virus (SIV). Behavioral testing after SIV inoculation was conducted in each group (-ethanol [EtOH]/-SIV, -EtOH/+SIV, +EtOH/-SIV, and +EtOH/+SIV) on days when alcohol was not administered to avoid a direct confound and on several occasions when ethanol or sucrose was administered as a probe of the effect of alcohol alone and the effect of caloric supplementation on the food-maintained tasks, respectively. RESULTS: During the days of the week when ethanol was not administered, little or no disruption was observed in either response rate or the percentage of errors (accuracy) across the different treatment groups. In contrast, behavioral testing during alcohol administration revealed that subjects in the various treatment groups had different susceptibilities to ethanol administration. As expected, a two-way ANOVA (ethanol condition, SIV condition) indicated there were significant main effects of ethanol on both response rate and percent errors in both behavioral tasks, but it also indicated there was a significant interaction between ethanol administration and SIV infection on the accuracy of responding in the acquisition (learning) task. In addition, the main effect of SIV on percent errors was in the performance task. CONCLUSIONS: The fact that alcohol administration in SIV-infected monkeys produced greater behavioral deficits than either alcohol or SIV alone further strengthens the supposition that IVs adversely affect neural substrates involved in cognition and that the adverse effects of many central nervous system drugs may be enhanced in IV-infected individuals.

Assessment of the nasopharyngeal bacterial flora of rhesus macaques: moraxella, Neisseria, haemophilus, and other genera.

The nasopharyngeal bacterial flora of healthy rhesus macaques was surveyed for the presence of Neisseria and Haemophilus species, as well as Moraxella catarrhalis. M. catarrhalis was found both in healthy rhesus macaques and in possibly immunocompromised rhesus macaques. Several Haemophilus spp. that are part of the normal nasopharyngeal bacterial flora of humans were found in many animals; these Haemophilus species included H. parahaemolyticus, H. segnis, and H. parainfluenzae. While Haemophilus influenzae was not identified, it is possible that the identification of H. influenzae types may have been thwarted by the growth of less fastidious species. A number of animals harbored Neisseria spp. such as N. sicca. However, Neisseria meningitidis was not found. In summary, it appears as though the rhesus macaque may be used as a model for M. catarrhalis infections. Moreover, in view of the susceptibility of macaques to organisms of the Haemophilus and Neisseria genera, it is possible that these animals may also accurately model nontypeable H. influenzae and N. meningitidis infections.