Composition of fecal microbiota of laboratory mice derived from Japanese commercial breeders using 16S rRNA gene clone libraries.

The fecal microbiota of six mice derived from three Japanese commercial breeders was analyzed by using 16S rRNA gene clone libraries to construct a database for analyzing the gut microbiota of laboratory mice. The 566 clones were obtained from the clone libraries generated from the fecal DNA samples derived from BALB/c, C57BL/6N, DBA/2 and ICR mice. Among these 566 clones, there were 446 unique 16S rRNA gene sequences. When grouped at the 98% similarity level, the 446 unique sequences consisted of 103 Clostridiales, 43 Bacteroidales, 5 Lactobacillus and 3 Erysipelotricaceae, as well as sequences from 11 other phyla.

Evaluation of E. ruminantium genes in DBA/2 mice as potential DNA vaccine candidates for control of heartwater.

Heartwater caused by the rickettsia Ehrlichia ruminantium (E. ruminantium) is an acute and fatal tick-borne disease of domestic and some wild ruminants. A user-friendly vaccine does not exist. We selected and tested nine genes of E. ruminantium for protection against challenge in a DBA/2 mouse model, in order to identify candidate genes for incorporation into a recombinant vaccine. Of the nine DNA vaccine constructs tested, four DNA constructs 14HWORF1/VR1012, 14HWORF2/VR1012, 27HWORF1/VR1012, and HSP58/VR1012 were not protective and were excluded from the study. The remaining five DNA constructs-MAP2/ VR1012, 1HWORF3/ VR1012, 4HWORF1/ VR1012, 18HWORF1/ VR1012, and 3GDORF3/ VR1012-offered partial protection against lethal challenge demonstrated by reduced mortalities compared to control groups. Protection was augmented when DNA primed mice were boosted with a respective homologous recombinant protein. Protection in these five groups was associated with the induction of cell-mediated or T helper 1 (Th1) type of immune responses characterized by the production of large amounts of interferon-gamma and interleukin-2 in in vitro proliferation assays using E. ruminantium antigens for stimulation. These responses were enhanced when the DNA-vaccinated DBA/2 mice were boosted with specific homologous recombinant protein vaccination. In a preliminary follow-up study, protection conferred by DNA vaccination with individual gene constructs was not enhanced when the protective constructs were administered in combination (including the map-1 gene of E. ruminantium). Further evaluation of these and other untested DNA constructs is necessary to optimize their expression in vivo in the presence of molecular adjuvants, such as the IFN-gamma gene, GM-CSF gene, IL-12 gene, and CpG motifs to fully evaluate their protective value.

Calcineurin controls growth, morphology, and pathogenicity in Aspergillus fumigatus.

Calcineurin is implicated in a myriad of human diseases as well as homeostasis and virulence in several major human pathogenic microorganisms. The fungus Aspergillus fumigatus is a leading cause of infectious death in the rapidly expanding immunocompromised patient population. Current antifungal treatments for invasive aspergillosis are often ineffective, and novel therapeutic approaches are urgently needed. We demonstrate that a mutant of A. fumigatus lacking the calcineurin A (cnaA) catalytic subunit exhibited defective hyphal morphology related to apical extension and polarized growth, which resulted in drastically decreased filamentation. The delta cnaA mutant lacked the extensive lattice of invading hyphae seen with the wild-type and complemented strains. Sporulation was also affected in the delta cnaA mutant, including morphological conidial defects with the absence of surface rodlets and the added presence of disjunctors creating long conidial chains. Infection with the delta cnaA mutant in several distinct animal models with different types of immunosuppression and inoculum delivery led to a profound attenuation of pathogenicity compared to infection with the wild-type and complemented strains. Lung tissue from animals infected with the delta cnaA mutant showed a complete absence of hyphae, in contrast to tissue from animals infected with the wild-type and complemented strains. Quantitative fungal burden and pulmonary infarct scoring confirmed these findings. Our results support the clinical observation that substantially decreasing fungal growth can prevent disease establishment and decrease mortality. Our findings reveal that calcineurin appears to play a globally conserved role in the virulence of several pathogenic fungi and yet plays specialized roles in each and can be an excellent target for therapeutic intervention.

A murine leukemia virus expressed in aged DBA/2 mice is derived by recombination of the Emv-3 locus and another endogenous gag sequence.

Although most inbred strains of mice contain endogenous retroviral sequences, these sequences are usually not capable of producing infectious retroviruses. In some cases, the retroviral sequences are small fragments of viral genomes. In a few cases the sequences for complete retroviruses exist, but contain small defects which prevent the production of infectious virus. While the ability of reversions of these defects to produce retroviruses has been studied by site-directed mutagenesis and chemical-mediated mutagenesis, the presence of spontaneously occurring reversions has not been completely evaluated. We characterized an infectious ecotropic retrovirus spontaneously expressed in aged DBA/2 mice, which carries the complete but defective Emv-3. Although this endogenously produced retrovirus was related to the endogenous Emv-3 sequence, it had undergone recombination with another retroviral sequence to correct the defect which resided in the gag of Emv-3. Thus, recombination of endogenous ecotropic retroviral sequences may be a mechanism to produce infectious retroviruses in adult animals that contributes to pathologic disease.

Endogenous nonecotropic proviruses mapped with oligonucleotide probes from the long terminal repeat region.

We characterized endogenous proviruses in C57BL/6J, DBA/2J, and C3H/HeJ mouse strains with oligonucleotide probes derived from long terminal repeat (LTR) sequences of three classes of nonecotropic murine leukemia virus. The segregation of proviral-host DNA junction fragments was followed in BXH and BXD recombinant inbred (RI) strain sets, and most fragments mapped readily to defined chromosomal regions. Most of the LTR fragments appear to correspond to proviruses mapped previously with oligonucleotide env region probes of the same viral class. At least 22 elements represent new proviral loci, no more than half of which may be solo LTRs, and an additional six may correspond to proviruses identified previously with less specific hybridization probes. Together with proviruses identified previously with env probes, the LTR probe-reactive elements represent the majority of endogenous murine leukemia proviruses in the mouse genome.

Detection of quantitative trait loci from frequency changes of marker alleles under selection.

A method was developed to estimate effects of quantitative trait loci (QTL) by maximum likelihood using information from changes of gene frequency at marker loci under selection, assuming an additive model of complete linkage between markers and QTL. The method was applied to data from 16 molecular and coat colour marker loci in mouse lines derived from the F2 of two inbred strains which were divergently selected on 6-week weight for 21 generations. In 4 regions of the genome, marker allele frequencies were more extreme than could be explained by sampling, implying selection at nearby QTL. An effect of about 0.5 standard deviations was located on chromosome 11, and accounted for nearly 10% of the genetic variance in the base population. QTL with effects as small as 0.2 phenotypic standard deviations could be detected. For typing of a given number of individuals, the power of detection of QTL is very high compared to, for example, analysis of an F2 population. The joint effects of linkage and selection were investigated by Monte Carlo simulation. Marker gene frequencies change little as a consequence of selection at a QTL unless the marker and QTL are less than about 20 cM apart.

Susceptibility to pristane-induced arthritis is altered with changes in bowel flora.

Pristane-induced arthritis (PIA) is unique among the animal arthritides in that a non-infectious, non-antigenic oil induces a chronic immune based arthritis with a prolonged delay between exposure to the inciting agent and development of the disease. Mice with pristane-induced arthritis have elevated T cell and humoral responses to the 65 kDa heat shock protein derived from Mycobacterium bovis (hsp65) and in common with several other models of autoimmune diseases the incidence of PIA is markedly suppressed by preimmunisation with hsp65 in Freund's incomplete adjuvant (Thompson et al. (1990) Eur. J. Immunol. 20, 2479). Recent studies have investigated how autoimmune reactions to heat shock proteins are involved in the development of arthritis. Arthritic CBA/Igb mice given pristane alone develop antibodies to both hsp65 and GroEl (bacterial 60 kDa heat shock proteins) and to hsp58 (the mammalian equivalent). Moreover, the splenic T cells of such mice proliferate vigorously in response to both bacterial and mammalian 60 kDa heat shock proteins. Remarkably, the anti-hsp65 antibody response in normal mice rises rapidly with age, directly correlating with the age related incidence of PIA. In addition, specific pathogen free mice (SPF) maintained in an isolator have negligible anti-hsp65 responses but these convert to positive responses if the animals are exposed to the open part of the animal facility (Thompson et al. (1992) Arthritis Rheum. 35, 139).(ABSTRACT TRUNCATED AT 250 WORDS)

Tumor necrosis factor-alpha mediates the early pathology in Salmonella infection of the gastrointestinal tract.

Salmonella infection of the intestinal tract results in damage to the gut epithelium. While it is generally believed that bacteria and/or bacterial products account for this pathology, the role of host factors has not been explored. Using a ligated intestinal loop model, we investigated whether tumor necrosis factor-alpha (TNF-alpha) could contribute to the tissue pathology associated with Salmonella infection. Intestinal segments infected with Salmonella typhimurium had high levels of fluid secretion as early as 6 h post-bacterial infection. At this time point, low levels of TNF activity were also present in the fluid obtained from infected segments. At 20 h post-infection, high levels of TNF activity were present in fluids obtained from infected intestinal segments and was characterized as TNF-alpha by neutralization experiments using rabbit antisera to TNF-alpha. TNF-alpha production was further verified by Northern blot analysis using RNA obtained from cells eluted from the infected intestinal segments. In contrast, no TNF activity was found in fluid obtained from intestinal segments challenged with cholera toxin, which induces fluid secretion with little to no inflammatory response. Double labeling by in situ hybridization and immunocytochemistry revealed that macrophages in the lamina propria were producing the TNF-alpha mRNA. To investigate what role TNF-alpha might play in Salmonella-induced inflammation, intestinal segments were injected with recombinant mouse TNF-alpha (rTNF-alpha) or mice were pretreated with antibody to TNF-alpha or a control antibody prior to Salmonella infection. The histological profile of intestinal segments injected with rTNF-alpha appeared identical to segments infected with S. typhimurium. Further, pathology was completely eliminated in infected mice pretreated with antibody to TNF-alpha. These results document the production of TNF-alpha in the intestinal tract following S. typhimurium infection and show that the early pathology induced by Salmonella infection of the gastrointestinal tract is mediated by immune mechanisms.

Use of DBA/2N mice in models of systemic candidiasis and pulmonary and systemic aspergillosis.

Mouse models of systemic candidiasis and pulmonary and systemic aspergillosis were established by using DBA/2N mice, which are known to be deficient in the C5 component of complement. In experiments comparing lethality in the respective models in DBA/2N versus outbred CFW mice, results showed that the 50% lethal dose values for the DBA/2N mice were 10- to 1,000-fold lower than those for the outbred mice, depending on the experiment. Additionally, onset of death was somewhat delayed for the DBA/2N mice. In the case of the pulmonary aspergillosis model, administration of cortisone acetate was necessary to ensure lethality after intranasal infection, but only a single dose was necessary.

Mousepox in inbred mice innately resistant or susceptible to lethal infection with ectromelia virus. V. Genetics of resistance to the Moscow strain.

The genetics of resistance to the Moscow strain of ectromelia virus was examined in crosses derived from resistant C57BL/6 (B6) and susceptible DBA/2 (D2) mice. Infection with 10(1) to 10(5) PFU of virus resulted in mortalities of 90 to 100% of D2, 0% of B6 and 0 to 3% of (B6 x D2) F1 mice by day 21. Among F1 x D2 backcross progeny, 49% of male and 18% of female mice died. Reciprocal backcrossing did not alter male or female mortality rates. These data are consistent with a single autosomal dominant gene controlling resistance to ectromelia in male mice and at least one additional dominant sex-limited gene controlling resistance in female mice. Fewer male F2 mice died than were predicted based on single-locus control and 32% of recombinant inbred (RI) strains derived from B6 and D2 progenitors expressed non-parental phenotypes. Therefore, additional resistance genes, not expressed in backcross mice, were apparently expressed in F2 mice and RI strains.

The endogenous mink cell focus-forming (MCF) gp70 linked to the Rmcf gene restricts MCF virus replication in vivo and provides partial resistance to erythroleukemia induced by Friend murine leukemia virus.

The Rmcf locus restricts the in vitro replication of mink cell focus-forming (MCF) viruses in cell cultures derived from mice carrying the resistance allele. Previously we reported that in cell cultures from first backcross progeny, this Rmcf-linked restriction segregates with the expression of an endogenous retroviral gp70 serologically related to that of MCF viruses. The current report details the results of genetic studies designed to examine the possible association of this endogenous gp70 with resistance of mice to Friend murine leukemia virus (F-MuLV)-induced erythroleukemia. This env gene segregates as a single dominant trait in (DBA/2 X IRW) X IRW progeny, in which the expression of the gene can be detected by serological techniques. Results indicated that the gp70- progeny developed leukemia at the same rate as the susceptible IRW parent, whereas the tempo of disease among the gp70+ progeny was significantly slower. However, the resistance mediated by this gene was only partial, since most of the gp70+ offspring eventually developed erythroleukemia when followed for 6 mo. This endogenous gp70 also segregated with a restriction to the expression of recombinant MCF viruses after infection with F-MuLV. Since in this study all unlinked genes segregated independently, this is direct evidence that MCF viruses participate in the induction of erythroleukemia.

Retroviral endogenous transcripts related to the envelope gene of Friend spleen focus-forming virus in normal mouse tissues.

Retroviral endogenous sequences related to the envelope (env) gene of Friend spleen focus forming virus (SFFV) and of mink cell focus forming viruses (MCF) are present in the genome of various mouse strains. We have examined the transcription of these SFFV/MCF-related sequences in normal tissues of two mouse strains, ICFW and DBA/2. Cytoplasmic Poly A+ RNAs of normal mouse tissues were analyzed by dot-blot and Northern blot hybridizations with a subcloned env SFFV DNA fragment (0.4 kbp BamH I-Sma I). In both mice, the level of SFFV/MCF env related transcripts was very low in bone marrows and spleens whereas it was high in kidneys. Intermediate levels of transcripts were observed in other tissues (thymus, liver and brain). In both mouse strains, the size of SFFV/MCF env related transcripts varied from one tissue to another. Some transcripts in DBA/2 mice were reminiscent of full-size viral message indicating an occasional expression of xenotropic/MCF endogenous virus in this low-leukemic strain. Sizes of the other SFFV/MCF related env transcripts were unusual, but were similar in both strains for each tissue studied. This last result suggests a tissue-specific transcription of endogenous sequences related to the SFFV/MCF env gene. A 1.8 kb SFFV/MCF env RNA was the major transcript in the tissues which expressed a high level of these env transcripts. Treatment of mice with phenylhydrazine which greatly stimulates erythroid differentiation in spleens increased the level of SFFV/MCF related env RNAs only in the spleens, suggesting a possible correlation between the SFFV/MCF env transcription and the stimulation of the erythroid spleen cells.

Spontaneous expression of C-type virus in DBA/2 mice is associated with an increased rate of mortality, independent of neoplastic disease.

Ecotropic C-type retroviruses isolated from both normal and dimethylbenzanthracene-treated DBA/2 mice could be classified into three major groups, Ea, Eb, and Ec, that differed in structure and biology. Weanling DBA/2 mice were generally free of viruses, but a fraction of adult individuals became virus positive and were apparently selectively associated with a high expression of the Eb viruses. Some of the ecotropic viruses from DBA/2 mice acted as exogenous pathogens. They caused viremia and a moderate incidence of leukemia when injected into C3H and ST/a mice. In addition, they caused an appreciable number of early deaths without signs of malignancy. To evaluate the natural role of the viruses, we studied the survival of spontaneously viremic and nonviremic DBA/2 mice. The viremic animals as a group were characterized by a significantly reduced life-span that was not related to neoplasia. These observations indicated that endogenous C-type retroviruses can be pathogenic without preselection of the host for disease. They also emphasize that endogenous viruses, like their exogenous counterparts, can have a broader pathogenic spectrum than normally appreciated.

A report of an outbreak of Salmonella oranienburg in a hybrid mouse colony.

Mice in a hybrid breeding colony developed symptoms compatible with a diagnosis of salmonellosis. Salmonella oranienburg was recovered from feces by conventional bacteriological techniques. Active salmonellosis appeared to be precipitated by the stress of pregnancy among breeder female BALB/c mice. Necropsy examination of the BALB/c breeder females revealed diarrhea, roughened hair coats, enlarged spleens, white foci in the liver, and skin abscesses. S. oranienburg was recovered from the spleen, cecum, oropharynx, uterus, and skin abscesses of affected mice. DBA/2N breeder males were asymptomatic and S. oranienburg was recovered from the cecum only. Breeding productivity indices dropped to unacceptable levels in rooms containing infected animals. Relatively normal production was observed among uninfected animals of the same strains in adjacent rooms, thus demonstrating the detrimental effect S. oranienburg had on this breeder colony.

Identification of ecotropic proviral sequences in high- and low-ecotropic-virus-producing mouse strains.

The arrangement of endogenous ecotropic retroviruses in selected high- and low-ecotropic-virus-producing mouse strains was examined by Southern blot hybridization analysis, using an ecotropic retrovirus-specific DNA probe. High-ecotropic-virus-producing mouse strains of the AKR family displayed heterogeneity with respect to the number of copies and the sites of insertion of endogenous ecotropic specific DNA. This diversity was seen even among individuals of the same AKR subline. Contrastingly, individuals within the same low-ecotropic-retrovirus-producing mouse strain showed no evidence of variability in their endogenous ecotropic proviral sequences. These results favored the hypothesis that germ line proviral reinsertion was responsible for the proviral sequence heterogeneity observed in high-ecotropic-virus-producing mouse strains.