Serological surveys of Corynebacterium kutscheri infection in mice and rats

Serological surveys of mice and rats naturally infected with Corynebacterium kutscheri were performed by examining serum samples collected from breeder and laboratory colonies between 1981 and 1983. Among 756 mice from 73 conventional colonies, only 4 animals (0.5%) from 3 colonies (4.1%) developed C. kutscheri antibody of 1:40 to 1:2, 560 titers. Three of them suffered from abscess caused by the organism. Regarding a titer of 1:40 or higher as reliably positive, 87 (13.0%) of 669 conventional rats or 20 (32.8%) of 61 colonies were found to be infected with the organism. The antibodies were detected in both types of animals older than 6 months of age. No lesions caused by C. kutscheri were found in almost all the rats examined. Germ-free and SPF mice and rats were all negative for antibody at 1:5 serum dilution.     

Time-course of Giardia muris infection in male and female immunocompetent mice

The time-course of acute Giardia muris infection was compared in male and female immunocompetent BALB/c mice that had not previously been exposed to the parasite. No sex-related difference was observed in the time-course of the infection in these mice. Sexually mature mice of both sexes excreted substantial numbers of G. muris cysts (greater than 10(4)/2 hr) over a longer period than did sexually immature mice.     

Sex differences in susceptibility of ICR mice to oral infection with Corynebacterium kutscheri.

Sex difference in susceptibility to oral infection with Corynebacterium (C.) kutscheri was experimentally studied in ICR mice. Immature (4-week-old) and adult (14-week-old) mice were inoculated with two infecting doses of C. kutscheri, and necropsied for bacteriological and serological survey 4 weeks after the bacterial infection. No macroscopic lesions at necropsy were demonstrated, except for one adult male given 10(9) bacteria. In immature mice, C. Kutscheri isolated from the oral cavity and cecum with FNC agar, were recovered in only 40.0% of female mice but in 90.0% of male mice given 10(6) bacteria (p < 0.05), and in only 55.6% of female mice but in 80.0% male mice given 10(8) bacteria. In adult mice given 10(9) bacteria, the organism were recovered in only 45.5% of female mice but in 90.9% of male mice (p < 0.05), furthermore, the mean number of organisms in the cecum of male mice harboring the organism was significantly higher than that in females (p < 0.01). Castration caused an increase in host resistance in adult male mice. These results indicated that ICR male mice were more susceptible than females, in terms of bacterial colonization in the cecum and the oral cavity, to oral infection with C. kutscheri.     

Increased susceptibility to Escherichia coli infection in mice pretreated with Corynebacterium parvum

The contribution of activated macrophages to protection against Escherichia coli was studied in mice treated intravenously with Corynebacterium parvum 7 days before infection. C. parvum-treated mice showed increased phagocytic activity and enhanced resistance to Listeria infection. In contrast, these mice showed increased susceptibility to a subsequent challenge with E. coli that correlated closely with a reduction in the LD50 of lipopolysaccharide (LPS) in these mice. The peritoneal macrophages obtained from C. parvum-treated mice had a strong ability to phagocytize and kill E. coli in in vitro experiments. A rapid decline in the number of bacteria in the liver of C. parvum-treated mice was observed in the early period of infection. However, the number of bacteria in liver and spleen increased progressively to a lethal dose from 6 hr after infection. At this time, a significant increase in beta-glucuronidase, a lysosomal acid hydrolase, was found in the serum of these mice. In vitro experiments revealed that the peritoneal macrophages from C. parvum-treated mice were highly susceptible to the cytotoxic effect of LPS after 6 hr of incubation with LPS. It is suggested that the hypersensitivity of activated macrophages to the cytotoxic effect of endotoxin derived from E. coli may be partly responsible for the increased susceptibility of C. parvum-treated mice to E. coli infection.     

Cell wall composition in Corynebacterium bovis and some other corynebacteria

The cell wall compositions of two strains of Corynebacterium bovis were found to differ: one contained lysine, rhamnose, mannose, and glucose, the other meso-alpha, epsilon, diaminopimelic acid (DAP), arabinose, galactose, and mannose. The walls of a strain of C. nephridii were characterized by l-DAP and galactose. Those of a strain of C. paurometabolum and of two strains of "lipophilic diphtheroids" contained meso-DAP, arabinose, galactose, and mannose as did walls of a reference strain of C. xerosis. The results are discussed in relation to the taxonomy of the organisms examined.     

Antimicrobial Susceptibility of Corynebacterium bovis Isolates from Immunodeficient Rodents

Corynebacterium bovis, the causative agent of hyperkeratotic dermatitis in immunodeficient mice, is a significant problem in preclinical oncology research. Infection results in lifelong skin colonization and a decrease in successful engraftment of patient-derived xenograft tumor models. The use of antimicrobial agents for C. bovis is controversial in light of reports of poor efficacy and the possibility of selection for resistant strains. The purpose of this study was to describe the antimicrobial susceptibilities of C. bovis isolates obtained exclusively from immunodeficient rodents in order to aid in antimicrobial dose determination. Between 1995 and 2018, 15 isolates were collected from 11 research institutions across the United States. Antimicrobial susceptibility testing was performed for 24 antimicrobials commonly used against gram-positive bacteria. Our results provide an updated understanding of the susceptibility profiles of rodent C. bovis isolates, indicating little variability between geographically and temporally distant isolates. These results will facilitate appropriate antimicrobial use to prevent and treat C. bovis infections in immunodeficient rodents.

Corynebacterium bovis is a gram-positive, facultatively anaerobic pleomorphic bacillus that infrequently causes infections in humans⁵ but is more clinically relevant in veterinary medicine. Veterinary interest in this bacterium originated in the dairy industry, where it causes subclinical mastitis in infected animals and is the most common Corynebacterium spp. isolated from infected udders. When present as a primary infection, C. bovis can cause decreases in milk quality with no significant decrease in milk yield.^9,12 Despite being considered a minor pathogen, the impact of C. bovis on milk quality remains economically important to the dairy industry.C. bovis was first recognized in the mid1970s in athymic nude mice with hyperkeratotic dermatitis, a condition that would later be termed ‘scaly skin disease.’⁶ Once genetically characterized in the mid1990s and confirmed to have an association with clinical disease, C. bovis emerged as an important pathogen of immunodeficient mice in the laboratory animals.⁷ Historically, C. bovis infections of research mice primarily occurred in athymic nude mice. However, as the number of transgenic immunodeficient strains has expanded, C. bovis is no longer considered an infection exclusively of athymic nude mice, as infections have been reported in immunodeficient and ‘immune-vague’ research rodents around the world.^{3,10,11,15,21}Antimicrobial susceptibility testing is used to identify the minimum inhibitory concentration (MIC) of specific antimicrobials that prevents the growth of an individual bacterial isolate in vitro. By including many isolates of the same organism into a test population, the MIC can be calculated that inhibits the growth of 50% (MIC₅₀) or 90% (MIC₉₀) of the isolates.²² MIC have been published for C. bovis isolates obtained from dairy cows.²⁵ In the dairy industry, dry cow therapy (the administration of antibiotics at the end of lactation) is highly effective at eliminating subclinical mastitis caused by Corynebacterium spp.¹ However, elimination of C. bovis from immunodeficient mouse populations is much more challenging.^15,17 To date, the dose of amoxicillin used to treat C. bovis-infected immunodeficient mice has been informed by MIC data from dairy cows isolates²⁵ and in vivo pharmacokinetic data in the form of blood plasma concentrations of amoxicillin administered in the drinking water.¹⁶ However, our group and others have demonstrated the reemergence of infection in immunodeficient mice after the discontinuation of antibiotic administration in a C. bovis-free environment. These findings suggest that the MIC for C. bovis isolates from mice may differ from that of cows.²Recently, the genomes of C. bovis isolates obtained from humans, cows, mice, and rats were sequenced. Subsequent genomic comparisons assessing the average nucleotide identity between isolates identified sequence divergence obtained from humans and cows as compared with isolates from rodents.⁴ In particular, the number of genomic islands and virulence factors were significantly higher in the rodent isolates than in the human and cow isolates. However, whether phenotypic changes in antimicrobial susceptibility accompany this genetic divergence is unknown. Considering the prior observations and new developments in our understanding of C. bovis across multiple species, the purpose of this study is to describe antimicrobial susceptibility profiles of C. bovis isolates obtained exclusively from immunodeficient rats and mice.     

The Nutritional Requirements and Biochemical Reactions of Corynebacterium bovis

S ummary . The cultural characteristics and biochemical reactions of 142 isolates of Corynebacterium bovis were examined and found to include the production of acid from glucose, fructose and glycerol, and the hydrolysis of urea. The reactions on lipolysis test media were equivocal, in part due to the inability of the organism to grow on some media, and also due to the doubtful validity of using Tween agar to test for lipase production. C. bovis demonstrated a requirement for serum, Tween 20 or 80, or egg yolk. In experiments carried out with fully defined liquid media this nutritional requirement was fulfilled by synthetic lecithin or Tween 80, or a mixture of both compounds.     

Impaired protection against Mycobacterium bovis bacillus Calmette-Guerin infection in IL-15-deficient mice

To investigate the potential role of endogenous IL-15 in mycobacterial infection, we examined protective immunity in IL-15-deficient (IL-15(-/-)) mice after infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG) or recombinant OVA-expressing BCG (rBCG-OVA). IL-15(-/-) mice exhibited an impaired protection in the lung on day 120 after BCG infection as assessed by bacterial growth. CD4(+) Th1 response capable of producing IFN-gamma was normally detected in spleen and lung of IL-15(-/-) mice on day 120 after infection. Although Ag-specific CD8 responses capable of producing IFN-gamma and exhibiting cytotoxic activity were detected in the lung on day 21 after infection with rBCG-OVA, the responses were severely impaired on days 70 and 120 in IL-15(-/-) mice. The degree of proliferation of Ag-specific CD8(+) T cells in IL-15(-/-) mice was similar to that in wild-type mice during the course of infection with rBCG-OVA, whereas sensitivity to apoptosis of Ag-specific CD8(+) T cells significantly increased in IL-15(-/-) mice. These results suggest that IL-15 plays an important role in the development of long-lasting protective immunity to BCG infection via sustaining CD8 responses in the lung.     

Duration of mouse hepatitis virus infection: studies in immunocompetent and chemically immunosuppressed mice

The duration of mouse hepatitis virus (MHV) infection was examined in mice inoculated intranasally with selected strains of MHV. Following inoculation with virulent MHV-JHM, genetically susceptible BALB/c mice and resistant CD1 mice had detectable virus in the brain at 1 month, but not later intervals up to 12 months. BALB/c mice infected with avirulent MHV-S or MHV-1 had no detectable virus in brains at 1 month or thereafter. Immunosuppression of BALB/c mice with treatment regimens of hydrocortisone acetate or cyclophosphamide at 1 and 2 months after infection with MHV-JHM did not activate detectable virus in liver or increase the prevalence or degree of brain infection. Immunosuppression with these drugs during the acute phase of MHV-JHM infection influenced MHV infection, based on virus quantification in livers, but timing of drug treatment relative to MHV infection was critical. Mice infected with MHV developed IgG serum antibody titers that persisted without decline for up to 1 year after infection. Antibody titers varied with mouse genotype and infecting virus. These studies, using intranasal inoculation, support the conclusions of others, using other routes of inoculation, that MHV infection is not persistent in adult, immunocompetent mice.     

Corynebacterium parvum- and Mycobacterium bovis Bacillus Calmette and Guerin-induced granuloma formation in mice lacking CD4 and CD8

Granuloma formation is a T-cell-dependent inflammatory response that is important in the host defense against intracellular bacteria. The role of CD4 and CD8 molecules in the development of Corynebacterium parvum- and Mycobacterium bovis Bacillus Calmette and Guerin (BCG)-induced granulomas was examined in CD4/CD8 knockout (KO) mice. CD4/CD8 KO mice developed a greater granulomatous response to heat-killed C. parvum and heat-killed BCG than did control mice. Thus, granuloma formation is not dependent upon the presence of CD4 and CD8. On the other hand, CD4/CD8 KO mice challenged with live BCG showed initially fewer and smaller granulomas but later more and larger granulomas than control mice. CD4/CD8 KO mice had a greater BCG load than control mice. The absence of CD4 and CD8 therefore impaired the host defense against infection with BCG. alphabeta T-cells were present in the granulomas of both CD4/CD8 KO and control mice in similar numbers. Also the production of IFN-gamma mRNA was similar in the two groups. In conclusion, CD4 and CD8 are not essential to the granulomatous response against C. parvum and BCG, but contribute to the host defense against live BCG infection.     

Interleukin-6 production in Mycobacterium bovis BCG-infected mice.

Mycobacterium bovis BCG and its subcellular components (bacterial extract, culture filtrate, purified protein derivative, and muramyl dipeptide MDP) are potent in vitro IL-6 inducers in spleen cell cultures from uninfected and BCG-infected BALB/c mice. Both plastic adherent and nonadherent spleen cells are capable of producing IL-6. Athymic nude mice produce more IL-6 than euthymic mice, suggesting that monocyte/macrophages are the main IL-6-producing cells in response to BCG. Finally, IL-6 production seems to be controlled to some extent by T lymphocytes, as down-regulation of CD4+ cells resulted in a marked increase in IL-6 production. Interferon-gamma does not seem to be involved in this regulation.     

Immunobiology of infection with recombinant vaccinia virus encoding murine IL-2. Mechanisms of rapid viral clearance in immunocompetent mice.

CD8+ cytotoxic T (Tc) lymphocytes mediate recovery from vaccinia virus (VV) infection. In mice, anti-VV Tc cells are detectable on or after day 3 after infection, and cytolytic activity peaks between days 5 and 6. A rVV encoding murine IL-2, VV-hemagglutinin (HA)-IL-2, was cleared more rapidly, compared with a control rVV, VV-HA-thymidine kinase (TK), from tissues of infected euthymic normal mice. The mechanism of VV-HA-IL-2 clearance was operative early in infection and correlated with an elevated NK cell response, before the induction of anti-VV Tc cell response. We have investigated the roles of NK cells, T cells, and IFN-gamma in the rapid clearance of VV-HA-IL-2, by using specific mAb. Depletion of NK cells with mAb significantly enhanced VV-HA-IL-2 but not VV-HA-TK titers 3 days after infection. NK cells alone could not account for rapid viral clearance, because VV-HA-IL-2 titers in NK cell-depleted mice were not comparable to VV-HA-TK titers. Treatment with a mAb to IFN-gamma completely abrogated the IL-2-induced mechanism(s) of VV-HA-IL-2 clearance, and titers of the IL-2-encoding virus were comparable to control virus titers. In addition, the elimination of CD4+ but not CD8+ T cells resulted in significant increases in VV-HA-IL-2 titers.     

Control of Mycobacterium bovis BCG infection with increased inflammation in TLR4-deficient mice

Live mycobacteria have been reported to signal through several pattern recognition receptors (PRR), among them toll-like receptor 4 (TLR4) and TLR2 in vitro. Here, we investigated the role of TLR4 in host resistance to Mycobacterium bovis (BCG) infection in vivo. In vitro, macrophages of TLR4 mutant C3H/HeJ mice infected with BCG expressed lower levels of TNF than controls, and TNF release was further decreased, although not completely absent, in the absence of TLR2. In vivo, TLR4 mutant C3H/HeJ and control C3H/HeOUJ mice were infected with BCG (2 x 10(6) CFU i.v.). Both TLR4 mutant and wild-type mice were able to control the infection and survived 8 months post-BCG infection. Macrophage activation with abundant acid-fast bacilli and expression of inducible nitric oxide synthase (iNOS) and MHC class II antigens was seen in both groups of mice. However, TLR4 mutant mice experienced an arrest of body weight gain and showed signs of increased inflammation, with persistent splenomegaly, increase in granuloma number and augmented neutrophil infiltration. Infection of TLR4-deficient mice with higher doses of BCG (1 and 3 x 10(7) CFU, i.v.) increased the inflammation in spleen and liver, associated with a transient, higher bacterial load in the liver. In summary, TLR4 mutant mice show normal macrophage recruitment and activation, granuloma formation and control of the BCG infection, but this is associated with persistent inflammation. Therefore, TLR4 signaling is not essential for early control of BCG infection, but it may have a critical function in fine tuning of inflammation during chronic mycobacterial infection.