Borrelia Host Adaptation Protein (BadP) Is Required for the Colonization of a Mammalian Host by the Agent of Lyme Disease.

Borrelia burgdorferi, the agent of Lyme disease (LD), uses host-derived signals to modulate gene expression during the vector and mammalian phases of infection. Microarray analysis of mutants lacking the Borrelia host adaptation regulator (BadR) revealed the downregulation of genes encoding enzymes whose role in the pathophysiology of B. burgdorferi is unknown. Immunoblot analysis of the badR mutants confirmed reduced levels of these enzymes, and one of these enzymes, encoded by bb0086, shares homology to prokaryotic magnesium chelatase and Lon-type proteases. The BB0086 levels in B. burgdorferi were higher under conditions mimicking those in fed ticks. Mutants lacking bb0086 had no apparent in vitro growth defect but were incapable of colonizing immunocompetent C3H/HeN or immunodeficient SCID mice. Immunoblot analysis revealed reduced levels of proteins critical for the adaptation of B. burgdorferi to the mammalian host, such as OspC, DbpA, and BBK32. Both RpoS and BosR, key regulators of gene expression in B. burgdorferi, were downregulated in the bb0086 mutants. Therefore, we designated BB0086 the Borrelia host adaptation protein (BadP). Unlike badP mutants, the control strains established infection in C3H/HeN mice at 4 days postinfection, indicating an early colonization defect in mutants due to reduced levels of the lipoproteins/regulators critical for initial stages of infection. However, badP mutants survived within dialysis membrane chambers (DMCs) implanted within the rat peritoneal cavity but, unlike the control strains, did not display complete switching of OspA to OspC, suggesting incomplete adaptation to the mammalian phase of infection. These findings have opened a novel regulatory mechanism which impacts the virulence potential of Bburgdorferi.

Segregation Lag in Polyploid Cells of the Pathogen Genus Borrelia: Implications for Antigenic Variation
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Relapsing fever agents like Borrelia hermsii undergo multiphasic antigenic variation that is attributable to spontaneous DNA non-reciprocal transpositions at a particular locus in the genome. This genetic switch results in a new protein being expressed on the cell surface, allowing cells with that phenotype to escape prevailing immunity. But the switch occurs in only one of several genomes in these spirochetes, and a newly-switched gene is effectively "recessive" until homozygosity is achieved. The longer that descendants of the switched cell expressed both old and new proteins, the longer this lineage risks neutralization by antibody to the old protein. We investigated the implications for antigenic variation of the phenotypic lag that polyploidy would confer on cells. We first experimentally determined the average genome copy number in daughter cells after division during mouse infection with B. hermsii strain HS1. We then applied discrete deterministic and stochastic simulations to predict outcomes when genomes were equably segregated either linearly, i.e. according to their position in one-dimensional arrays, or randomly partitioned, as for a sphere. Linear segregation replication provided for a lag in achievement of homozygosity that was significantly shorter than could be achieved under the random segregation condition. For cells with 16 genomes, this would be a 4-generation lag. A model incorporating the immune response and evolved matrices of switch rates indicated a greater fitness for polyploid over monoploid bacteria in terms of duration of infection.

Genome Report: Identification and Validation of Antigenic Proteins from Pajaroellobacter abortibovis Using De Novo Genome Sequence Assembly and Reverse Vaccinology.

Epizootic bovine abortion (EBA), or "foothill abortion," is the leading cause of beef cattle abortion in California and has also been reported in Nevada and Oregon. In the 1970s, the soft-shelled tick Ornithodoros coriaceus, or "pajaroello tick," was confirmed as the disease-transmitting vector. In 2005, a novel Deltaproteobacterium was discovered as the etiologic agent of EBA (aoEBA), recently named Pajaroellobacter abortibovis This organism cannot be grown in culture using traditional microbiological techniques; it can only be grown in experimentally-infected severe combined immunodeficient (SCID) mice. The objectives of this study were to perform a de novo genome assembly for P. abortibovis and identify and validate potential antigenic proteins as candidates for future recombinant vaccine development. DNA and RNA were extracted from spleen tissue collected from experimentally-infected SCID mice following exposure to P. abortibovis This combination of mouse and bacterial DNA was sequenced and aligned to the mouse genome. Mouse sequences were subtracted from the sequence pool and the remaining sequences were de novo assembled at 50x coverage into a 1.82 Mbp complete closed circular Deltaproteobacterial genome containing 2250 putative protein-coding sequences. Phylogenetic analysis of P. abortibovis predicts that this bacterium is most closely related to the organisms of the order Myxococcales, referred to as Myxobacteria. In silico prediction of vaccine candidates was performed using a reverse vaccinology approach resulting in the identification and ranking of the top 10 candidate proteins that are likely to be antigenic. Immunologic testing of these candidate proteins confirmed antigenicity of seven of the nine expressed protein candidates using serum from P. abortibovis immunized mice.

Foxp3(+) T cells regulate immunoglobulin a selection and facilitate diversification of bacterial species responsible for immune homeostasis.

Foxp3(+) T cells play a critical role for the maintenance of immune tolerance. Here we show that in mice, Foxp3(+) T cells contributed to diversification of gut microbiota, particularly of species belonging to Firmicutes. The control of indigenous bacteria by Foxp3(+) T cells involved regulatory functions both outside and inside germinal centers (GCs), consisting of suppression of inflammation and regulation of immunoglobulin A (IgA) selection in Peyer's patches, respectively. Diversified and selected IgAs contributed to maintenance of diversified and balanced microbiota, which in turn facilitated the expansion of Foxp3(+) T cells, induction of GCs, and IgA responses in the gut through a symbiotic regulatory loop. Thus, the adaptive immune system, through cellular and molecular components that are required for immune tolerance and through the diversification as well as selection of antibody repertoire, mediates host-microbial symbiosis by controlling the richness and balance of bacterial communities required for homeostasis.

Pathogenicity of Pasteurella pneumotropica in immunodeficient NOD/ShiJic-scid/Jcl and immunocompetent Crlj:CD1 (ICR) mice.

Pasteurella pneumotropica is an opportunistic pathogen in rodents. Natural infection in immunodeficient animals suggests that immunodeficiency is a major factor in P. pneumotropica pathogenesis. To understand this process, we performed clinical, pathological and bacteriological studies of immunodeficient NOD/ShiJic-scid/Jcl and immunocompetent Crlj:CD1 (ICR) mice experimentally infected with P. pneumotropica ATCC 35149. From 14 days postinoculation, some of P. pneumotropica-infected NOD/ShiJic-scid/Jcl mice developed clinical signs of weight loss. Three of 10 P. pneumotropica-infected NOD/ShiJic-scid/Jcl mice developed clinical signs of depression, ruffled coat, and weight loss and died at 27, 34, and 59 days postinoculation. At 35 days postinoculation, almost all P. pneumotropica-infected NOD/ShiJic-scid/Jcl mice had lung abscesses. The bacteria were isolated from the upper and lower respiratory tracts, including the lungs, and blood. In contrast, P. pneumotropica-infected ICR mice exhibited no clinical signs or lesions. The bacteria were isolated from the upper, but not the lower respiratory tracts. We developed an animal model for understanding host interactions with P. pneumotropica.

Coxiella burnetii infection in C.B-17 scid-bg mice xenotransplanted with fetal bovine tissue.

Two from a group of approximately 50 C.B-17 scid-bg mice were examined because of lethargy, dehydration, and rough coat. Three months prior to development of clinical signs of disease, mice of this study had been surgically implanted with fetal bovine liver, thymus, and lymph node. At necropsy, marked splenomegaly and mild hepatomegaly were observed in both animals. Large areas of necrosis and inflammation, with associated intracytoplasmic granular basophilic inclusions, were observed in histologic sections of multiple organs. Aerobic and anaerobic culturing of the liver yielded negative results. Six months after the initial case, four more reconstituted scid-bg mice from a different fetal donor had identical clinical, gross, and histologic signs of disease. To determine whether the basophilic inclusions represented an infective agent, 4-month-old immune-naive C.B-17 scid-bg mice were inoculated intraperitoneally with a liver and spleen homogenate from an affected mouse. Two weeks after inoculation, mice developed clinical signs of disease and lesions identical to those seen in the signal mice. On ultrastructural examination of the liver, pleomorphic bacteria were found in large cytoplasmic vacuoles of hepatocytes. Bacterial DNA was amplified from the liver, using primers that amplify a segment of the 16S rRNA gene from many bacterial species. Sequencing of the polymerase chain reaction (PCR) product revealed gene sequence identical to that of Coxiella burnetii, the agent of Q-fever. These results highlight the need to consider infective agents of the donor species when working with xenografted animals.

Helicobacter bilis/Helicobacter rodentium co-infection associated with diarrhea in a colony of scid mice.

An outbreak of diarrhea spanning 3 months occurred in a breeding colony of scid/Trp53 knockout mice. Approximately a third of the 150 mice were clinically affected, with signs ranging from mucoid or watery diarrhea to severe hemorrhagic diarrhea with mortality. Helicobacter bilis and the newly recognized urease-negative organism H. rodentium were isolated from microaerobic culture of feces or cecal specimens from affected mice. Dual infection with H. bilis and H. rodentium were confirmed by culture and polymerase chain reaction (PCR) in several animals. Both Helicobacter species rapidly colonized immunocompetent sentinel mice exposed to bedding from cages containing affected mice, but the sentinel remained asymptomatic. Mice with diarrhea had multifocal to segmental proliferative typhlitis, colitis, and proctitis. Several affected mice had multifocal mucosal necrosis with a few focal ulcers in the cecum, colon, and rectum. Mice with diarrhea were treated with antibiotic food wafers (1.5 mg of amoxicillin, 0.69 mg of metronidazole, and 0.185 mg of bismuth/mouse per day) previously shown to eradicate H. hepaticus in immunocompetent mice. Antibiotic treatment resulted in resolution of diarrhea, but not eradication of H. bilis and H. rodentium; mice continued to have positive PCR results after a 2-week treatment regimen, and clinical signs of diarrhea returned in some mice when treatment was suspended. To the authors' knowledge, this is the first report of natural infection with either H. bilis and/or H. rodentium causing acute diarrheal disease and suggests that H. bilis and/or H. rodentium can be an important pathogen for scid mice.

Isolation of Enterococcus durans and Pseudomonas aeruginosa in a scid mouse colony.

In a colony of severe combined immunodeficiency (SCID) mice conspicuously altered behavioural characteristics were observed: hunched position, apathy, dullness, short breath, bristled fur, emaciation, circling movements around their longitudinal axis and oblique head posture. This was most common in pregnant and lactating animals and also observed in 4 mice after experimental treatment. Pseudomonas aeruginosa, serotype P1 and Enterococcus durans, serotype D were isolated from various organs and from the middle ear. On autopsy, the mice showed signs of focal pericarditis and thickened liver capsules. The histological examination of the liver revealed mild, focal accumulations of mononuclear cells. In addition, it was observed that SCID mice with signs of this disease did not allow human peripheral blood mononuclear cells to engraft.

Translocation of bacteria from the gastrointestinal tract in immunodeficient mice.

Host defence mechanisms associated with the inhibition of translocation of bacteria from the gastrointestinal (GI) tract were investigated in SCID and beige mice after decontamination with oral antibiotics and colonization with Escherichia coli C25. SCID mice, which have impaired T and B cell function, tended to have a greater incidence of bacterial translocation from the GI tract up to 7 days after inoculation compared with controls. However, after 7 days both SCID and controls cleared the E. coli C25 from the liver, spleen, blood and peritoneal cavity. Beige mice, with impaired NK cell and polymorphonuclear leukocyte function, were not able to clear the inoculated bacteria from their liver by 14 days after inoculation although the controls were cleared by 7 days. Numbers of bacteria in the mesenteric lymph nodes (MLN) of beige mice did not decrease significantly by 14 days after inoculation, whereas numbers in SCID mice decreased markedly within 7 days. These results suggest that defence mechanisms other than T and B cell function are important in the inhibition of systemic infection from the GI tract.

The scid mouse as an experimental model for the evaluation of anti-Pneumocystis carinii therapy.

The usefulness of scid mice bearing endogenous Pneumocystis carinii infection as a model for experimental chemotherapy was examined using standard compounds known to be effective against P. carinii. Trimethoprim/sulphamethoxazole was able to reduce pulmonary P. carinii cysts in a dose-dependent manner within the dose range studied (10/50 to 100/500 TMP/SMX mg/kg/d, bd, po, 5 days per week for 30 treatments). However, alterations in associated symptoms of infection (reduced body weight, increased lung weight, increased blood leucocytes and erythrocytes), was apparently not linearly dose-dependent. Blood and lung lavage fluid levels of sulphamethoxazole one hour post administration of trimethoprim/sulphamethoxazole was dose-dependent, but not linear with dose, and was apparently correlated to cyst reduction; trimethoprim was below the limit of detection at this time. Treatment of mice with 100/500 mg/kg/day trimethoprim/sulphamethoxazole required 2 weeks (bd for 10 days of treatment) before changes in indices of infection became significant. Pentamidine (20 mg/kg, sc, three times per week for 3 weeks) was nearly as effective as high-dose trimethoprim/sulphamethoxazole in reducing cysts, whereas lower doses were ineffective. Despite being unable to reduce pulmonary P. carinii infection, even low doses of pentamidine (6 or 2 mg/kg, sc, three times per week for 3 weeks) were able to reduce lung weights and blood leucocyte levels. This model of pulmonary P. carinii infections is amenable to chemotherapeutic intervention in an apparently dose-dependent fashion, and can be used to evaluate the capacity of compounds to eradicate P. carinii and resolve signs of infection.

Latency, without persistence, of murine cytomegalovirus in the spleen and kidney.

It is not known if murine cytomegalovirus (MCMV) establishes a state of molecular latency independent of low-level persistent infection. The presence of low levels of infectious MCMV distinguishes persistence from molecular latency. Thus, the distinction between persistence and latency has depended on the sensitivity of plaque assays for detecting low levels of infectious virus in tissue of previously infected mice. To determine whether MCMV establishes molecular latency or remains persistent, we developed two assays for detecting low levels of MCMV in tissue. Using prolonged in vitro culture of virus with either mouse embryonic fibroblasts or the murine 3T12 fibroblast cell line, we reproducibly detected a single PFU of MCMV. Inclusion of undiluted sonicated tissue in this assay decreased sensitivity by up to 100-fold. However, sensitivity was improved to 1 PFU of MCMV when sonicated tissue was appropriately diluted. Severe combined immunodeficient (SCID) mice were also used to detect MCMV in sonicated tissue. Infection of SCID mice with a single PFU of MCMV killed two of eight SCID mice, and the 50% lethal dose of MCMV in SCID mice was 2 to 3 PFU. Applying these two methods, we detected infectious virus in 0 of 34 spleens, 1 of 34 kidneys, and 0 of 37 salivary glands from latently infected mice. Spleens and kidneys assessed for persistent virus contained MCMV DNA by PCR and reactivated after 10 to 50 days in explant cultures. Latently infected kidney cells reactivated after adoptive transfer to SCID mice. Quantitation of the MCMV genome by PCR showed that latently infected spleens without detectable infectious MCMV contained about 3,000,000 copies of the MCMV genome. These results demonstrate that MCMV latency in spleen and kidney exists in the absence of low-level persistent infection. Use of assays with defined sensitivity for detection of MCMV in tissue provides a basis for evaluation of cytomegalovirus gene expression in the spleen and kidney during molecular latency.

SCID mouse spleen does not support scrapie agent replication.

BALB/c and severe combined immunodeficiency (SCID) mice were inoculated intracerebrally or intraperitoneally with scrapie agent strain ME7 to examine the role of functional lymphocytes and follicular dendritic cells in splenic infectivity and PrPSc accumulation. Intracerebrally inoculated BALB/c and SCID mice developed the clinical signs and microscopic lesions characteristic of scrapie. Spleens from terminally affected BALB/c mice contained PrPSc which was detectable by immunoblot analysis; SCID mouse spleens did not contain detectable PrPSc. SCID mouse spleens collected during the first 90 days after intraperitoneal infection contained neither infectivity nor PrPSc.

Isolation of Acinetobacter calcoaceticus from the gastrointestinal tracts of SCID mice.

Acinetobacter calcoaceticus colonization was observed in the gastrointestinal tracts of C.-B17-scid/scid (SCID) mice, while it was not observed in C.B17-scid/+ and C.B17-+/+ mice with normal immunity housed under the same conditions. A. calcoaceticus and other viable enteric bacteria were not isolated from any organs other than gastrointestinal tract in SCID mice. The mice colonized with this organism were apparently healthy and no significant visceral lesion was observed.

Granuloma formation in severe combined immunodeficient (SCID) mice in response to progressive BCG infection. Tendency not to form granulomas in the lung is associated with faster bacterial growth in this organ.

Intravenous inoculation of Mycobacterium bovis, strain bacillus Calmette-Guerin caused infection in the lungs, livers, and spleens of severe combined immunodeficient (SCID) mice and in the same organs in immunocompetent co-isogenic C.B17 mice. However, whereas infection in the latter mice was stabilized and partly resolved, it was progressive in SCID mice and eventually lethal, with the most rapid bacterial growth occurring in the lungs. Histological examination of infected organs showed that well-developed, compact epithelioid granulomas formed at sites of bacterial multiplication in livers, spleens, and lungs of C.B17 mice. Granulomas also formed in the livers and spleens of SCID mice, despite their inability to generate immunity. However, in the lungs of SCID mice, bacillus Calmette-Guerin was regionally distributed mostly in isolated alveolar macrophages and in aggregates of macrophages resembling small granulomas. The possibility that this tendency not to form granulomas in the lung is the reason for the more rapid growth of bacillus Calmette-Guerin in this organ is discussed.

Spontaneous and experimental infections in scid and scid/beige mice.

Severe combined immunodeficient (scid) mice are valuable animals to study a variety of physiologic and disease processes. Their capacity to support multiple tissue xenografts permits these mice to be used as intermediate models for host-specific, fastidious organisms for which a small animal model has not been available previously. However, because they are unable to mount a normal immune response, they are very susceptible to a variety of primary and opportunistic microbial pathogens. Fatal, naturally occurring infections with bacteria such as Proteus mirabilis, Streptococcus viridans, and Escherichia coli have been observed. In addition, based on observations after experimental or naturally occurring viral infections, scid and scid/beige mice have been shown to be very susceptible to infections with viruses such as mouse hepatitis virus, Sendai virus, and murine respiratory virus, with resulting mortality. Of the parasitic infections, Pneumocystis carinii is a relatively common contaminant of the respiratory tracts of scid mice and may complicate research projects, particularly experimental respiratory tract infections. In view of the enhanced susceptibility of these mice to infections of this type, it is essential that they be housed under optimal conditions, which include implementing stringent management practices and a functional barrier system.

Severe pulmonary pneumocystosis in simian acquired immunodeficiency syndrome induced by simian immunodeficiency virus: its characterization by the polymerase-chain-reaction method and failure of experimental transmission to immunodeficient animals.

Pneumocystis carinii (Pc) infection was observed in three of five rhesus monkeys infected with simian immunodeficiency virus (SIVmac251). They showed severe symptoms similar to those associated with human acquired immunodeficiency syndrome (AIDS). Histopathology revealed severe pulmonary pneumocystosis in one of three Pc-positive monkeys, and anti-Pc antibodies were detected in sera from two of the three monkeys. Localization of Pc organisms in various organs of the monkeys was examined by the polymerase-chain-reaction (PCR) method, and Pc-specific bands of DNA amplification were detected in the liver, kidney, spleen, adrenal gland, testis, brain, and other organs examined, but no Pc organism was found in these organs by histopathologic examination. These results suggest that the activation of a latent infection of Pc occurs in SIV-infected rhesus monkeys as well as in human AIDS. Experimental transmission of Pc derived from a simian was attempted in severe combined immunodeficiency (SCID) mice and athymic nude (rnu/rnu, F344) rats. These animals were inoculated intranasally with 10(4) Pc cysts, but neither histopathologic changes nor Pc organisms were detected in SCID mice at 4 months after inoculation or in nude rats at 2 months postinoculation, suggesting that simian Pc is species-specific.

Experimental mycoses in congenitally immunodeficient rodents.

Congenitally immunodeficient rodents are playing a major role in clarifying host defense mechanisms and elucidating the virulence factors of a variety of mycotic agents. These murine models are also being used to study the histopathology and chemotherapy of a variety of fungal infections. Recent development of immunodeficient transgenic mice will make these murine models even more valuable for studies on the diagnosis, pathogenesis, immunity, and chemotherapy of fungal infections.