Detection of Corynebacterium kutscheri in the faeces of subclinically infected mice

Mice were infected experimentally and subclinically with Corynebacterium kutscheri to recover the organism from mice faeces. The faeces were then cultured using selective furazolidone-nalidixic acid-colimycin agar. The number of C. kutscheri per gram of fresh faeces varied from mouse to mouse, but once established in the intestine, the organism was excreted in the faeces for at least five months. Viable bacteria were detected in most of the faecal samples, including those stored in the animal room for five days. The number of organisms in the stored faeces decreased gradually but did not differ significantly from those in the fresh faeces until they had been stored for more than three days. Many infected mice excreted between 10(4.77) and 10(5.37) colony forming units (CFU) of C. kutscheri per day in their faeces, and one mouse even excreted 10(3.74) CFU at eight weeks postinfection. These values showed little daily variation. Our present study showed that subclinically infected mice discharged the organism continuously and persistently in their faeces. Therefore, faecal samples would be useful for monitoring infection with C. kutscheri in living mice in a manner that is not stressful for the animals.     

Isolation of Corynebacterium kutscheri from aged Syrian hamsters (Mesocricetus auratus).

Corynebacterium kutscheri was isolated from the oral cavities of 12 male Syrian hamsters (Mesocricetus auratus) which were about 12 months old. At 1, 5, and 9 months after initial isolation of C. kutscheri from the oral cavity, hamsters were euthanatized, and attempts were made to culture C. kutscheri from 13 additional sites. Corynebacterium kutscheri was isolated from nine hamsters, and regardless of the hamsters' ages, the organisms were most frequently isolated from the oral cavity (100%), esophagus (100%), cecal content (100%), and colon and rectum (88.9%). Isolation rates in the nasal cavity were 66.7%, followed by 55.5% in the trachea and 33.3% in the submaxillary lymph nodes. The number of the organisms found in the submaxillary lymph nodes and esophagus was 10(3) to 10(4) CFU/g. The number found in the cecal content and in the colon and rectum was 10(2) to 10(5) CFU/g. The organisms were not isolated from lung, stomach, kidney, spleen, and mesenteric lymph node tissues. The hamsters had neither clinical signs nor lesions. However, 7 of 12 animals had low agglutinating antibody titers. The Syrian hamster can therefore be an asymptomatic carrier of C. kutscheri.     

Selective medium for Corynebacterium kutscheri and localization of the organism in mice and rats

A new selective medium for isolation of Corynebacterium kutscheri (CK) from animals suffering subclinical infection was made by adding furazolidone, nalidixic acid and corimycin to the heart infusion agar base, this being named FNC agar. The FNC agar inhibited the growth of gram-negative rods, Pseudomonas aeruginosa, Proteus sp. and gram-positive cocci but did not affect the growth of CK. When this medium was used to isolate CK from the oral cavity and cecal contents of mice and rats, two of 6 conventional mouse colonies and three of 8 conventional rat colonies were found to be infected, with isolation of the organism from 19 mice and 12 rats in total. The animals showed neither clinical signs nor lesions, but the antibody was positive in 11 mice and 10 rats. In mice and rats inoculated orally with 4 x 10(8) and 1 x 10(9) organisms, respectively, CK was isolated for 20 weeks post-inoculation by use FNC agar. The isolation rate of the organism was the highest in the oral cavities of both inoculated mice and rats, and also in the submaxillary lymph nodes of the inoculated rats. The organism was also recovered from the cecal contents of more than half of the inoculated mice and rats. Thus, it was considered that FNC agar was useful in isolating CK from the oral cavity and cecal contents of mice and rats with subclinical infection of the organism.     

Pathological studies on corynebacterial ulcerative entero-colitis in rats treated with ACTH (author's transl)]

Ulcerative entero-colitis was developed in 6-week-old male Sprague-Dawley rats treated daily with ACTH (4 mg/kg. s. c.) as well as necrotic purulent lesions in liver, kidney, lung or heart. Incidence of ulcerative lesions was 6.3% in Farm-A rats and 56% in Farm-B rats. Although ulcerative lesions were mostly observed in cecum, the similar lesions were also detected in distal ileum or proximal colon in some cases. Histologically, the lesions were characterized by focal necrosis demarcated from surrounding normal tissue containing a number of clumps of bacteria and cellular debris. Bacteriological examination revealed that provocation of Coryne-bacterium kutscheri by ACTH-treatment resulted in appearance of the lesions. By means of intravenous or intraperitoneal inoculation with the strain isolated from lesion similar lesions were produced in the cecum of inoculated rats under the ACTH-treatment.     

Detection of Corynebacterium kutscheri from the oral cavity of rats.

A simple and useful method for the detection of C. kutscheri from the oral cavity of living rats was devised. In 10 sacrificed rats from two naturally and subclinically infected conventional colonies, 10(4.28) or 10(3.84) CFU/ml C. kutscheri were isolated from upper incisor swab extractions, while 10(1.38) or 10(1.58) and < 10 or 10(1.56) CFU/ml from the upper soft palate and pharynx, respectively. In another survey with 26 living animals, which were reared on the same rack, organisms were detected from the upper incisor and gingival swabs in 15 of 26 rats (57.7%). The results were reproducible at a second survey 10 days later. No organisms were isolated from any sites of the orally negative rats. These results indicated that culture of swab specimens from the upper incisors and gingivae of incisors is useful for the detection of C. kutscheri infection in living rats.     

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.     

Experimental Corynebacterium kutscheri infection in rats: bacteriology and serology

In a preliminary study, hydrocortisone-treated rats developed pseudotuberculosis when challenged with 6.2 X 10(5) to 3.1 X 10(7) colony forming units of Corynebacterium kutscheri by intranasal, intragastric, or subcutaneous inoculation. Oronasal exposure was selected as a likely natural route to further study inapparent infection. In Study 1, 50 rats received 1.2 X 10(5) colony forming units and various tissues were cultured at intervals to 12 weeks post-inoculation. At each interval, C. kutscheri was regularly isolated from submaxillary lymph nodes, but isolation was sporadic from other sites. In Study 2, 17 out of 21 rats given 1.2 X 10(5) colony forming units and killed weekly for 6 weeks had 2.0 X 10(2) to 1.8 X 10(5) colony forming units of C. kutscheri in oral washes, and 16 rats had 2.0 X 10(2) to 1.0 X 10(5) colony forming units in submaxillary lymph nodes, Serum antibody to C. kutscheri using both microagglutination and indirect immunofluorescence was first detected in some rats by 2 weeks, and in all rats at subsequent intervals. There was a significant (P less than 0.001) positive correlation (r = 0.93) between serum antibody titers and the duration of infection.     

Metronidazole-induced susceptibility of adult mice to intestinalClostridium botulinum colonization

Clostridium botulinum type A spore challenges were given orogastrically to adult mice that had been treated with metronidazole. The organism multiplied in the intestinal tract for up to five days if it was administered 9–48 h after the last dose of metronidazole. The known antibacterial spectrum of metronidazole indicated that obligate anaerobic bacteria are important in the natural resistance of adult mice to colonization byC. botulinum, but treatment with the antimicrobial agent did not reduce the number of these bacteria in the cecum and colon.     

Latent infection with Corynebacterium kutscheri in mice after peroral inoculation and its provocation by cortisone (author's transl)]

Latent infection with Corynebacterium kutscheri in mice and its provocation by cortisone were studied with a rifampicin-resistant strain of the organism. Mice having been infected perorally began to excrete the organisms in feces within 6 hours, and most of them were found to be carrying the organisms in the intestine, especially in the cecum even 90 days after infection. In such state of latency, however, no organisms were detected in other main organs, and neither visible lesions nor serum agglutinin was detectable. The latent infection with excretion of the organisms in feces after peroral infection was shown to become overt and fatal by cortisone treatment made even 90 days after infection. In infected mice excreting no organisms in feces and having bites on their skin, the wounds became severe ulcers after cortisone treatment resulting in septicemia.     

Abnormal glucose metabolism in murine cytomegalovirus (MCMV)-infected mice

Five strains of male and female mice were infected with salivary gland-passaged murine cytomegalovirus (SG-MCMV) intraperitoneally. Although none of them became hyperglycemic, 13-30% of BALB/c male and female, ICR/Slc male and female and C57BL/6J male mice had abnormal values in glucose tolerance tests (GTT) 14 to 30 day post infection. Serum insulin levels in BALB/c male mice were low and infective virus was detected in the pancreas during the acute phase of infection. Histopathology showed mild edematous changes with inflammatory cell infiltrations in the pancreas. Viral antigen was located in acinar cells and occasionally in beta cells by an immunofluorescence test.     

Oncospheres of Taenia solium and T. saginata asiatica develop into metacestodes in normal and immunosuppressed mice.

Normal and immunosuppressed mice were infected with oncospheres of Taenia saginata asiatica and T. solium. Although normal ICR mice were not susceptible to these two parasites, cysticerci were recovered from the immunosuppressed ones following venous injection. For T. s. asiatica, immunosuppressed ICR mice had an infection rate of 12.5% and six cysticerci of this parasite were recovered from three males. After injection of T. solium oncospheres, a high infection rate of 57% was obtained and 23 cysticerci were collected from 13 male immunosuppressed ICR mice. The immunosuppressed C57 mice had the highest infection rate (100%) and cysticercus recovery rate (2.4%) for T. solium. The infection rate and cysticercus recovery rate in six normal C57 mice were 40% and 3% respectively. The immunosuppressed ICR, Balb/c and C3H mice were also susceptible to T. s. asiatica.     

The effect of selected viruses on Corynebacterium kutscheri infection in rats

The influence of selected viral pathogens on rats that were previously infected with Corynebacterium kutscheri was investigated. A series of three separate experiments were performed to test the effect of sialodacryoadenitis virus, Sendai virus and rat virus. In each experiment, weanling rats were divided into three groups (C. kutscheri-inoculated, virus-inoculated and C. kutscheri plus virus-inoculated). Two groups were inoculated oronasally with C. kutscheri to establish subclinical infections. Two weeks later, two groups were inoculated intranasally with virus. At 5 weeks, the prevalence of C. kutscheri recovery from oral cavity and submaxillary lymph node and the prevalence of overt pseudotuberculosis was compared between treatment groups. Seroconversion of rats to C. kutscheri was measured by microagglutination and viruses by indirect immunofluorescence assays. Infection of rats with sialodacryoadenitis virus, Sendai virus or rat virus had no discernable effect on C. kutscheri-infected rats.     

Heligmosomoides polygyrus (=Nematospiroides dubius): the development of self-cure and/or protection in several strains of mice.

Strains of outbred (ICR/CD1 and S--W) and inbred (BALB/C and C57BL/6) mice vaccinated subcutaneously (SQ) with 500, 1,000, or 2,000 exsheathed Heligmosomoides polygyrus larvae developed varying levels of protection upon subsequent oral challenge with larvae. In contrast, the inbred C3H/HEJ strain failed to develop protection at any dosage level tested. ICR/CD1 mice vaccinated intraperitoneally with exsheathed larvae developed a high level of resistance but exhibited extensive adhesions of the viscera. When ensheathed larvae were used for vaccination, ICR/CD1 mice developed a moderate level of protection; but 1% of the vaccine dose was recovered in the intestine as adult stages. Both the inbred and outbred strains given multiple oral infections developed a protection response similar to that strain's response following parenteral vaccination. The specificity of this protection was demonstrated using various complex foreign antigens. In contrast, the self-cure response was observed only in the S--W strain.     

Salmonella ochiogu: experimental infection of laboratory mice and oxytetracycline therapy

The oral infection of laboratory mice with 10(8) colony-forming units of viable Salmonella ochiogu bacteria resulted in clinical salmonellosis and death in 10 out of 45 of the mice (22%). None of the mice treated with oxytetracycline died. Infection in susceptible mice was characterized by septicaemia, respiratory involvement and mild enteritis. The organism was shed in the faeces from the first day after infection until day 30, and cultures from viscera showed systemic dissemination. S. ochiogu was recovered from the faeces of mice treated with oxytetracycline between days 1 and 9 post infection.     

Age and strain dependence of O6-methylguanine DNA methyltransferase activity in mice

The activity of the DNA repair protein O6-methylguanine DNA methyltransferase (MT) was compared in liver extracts from female ICR and male C57BL/6 mice at various ages (3-130 weeks old). Similar patterns of overall enzyme activity were observed in both strains with O6-MT activity being relatively low in young mice (3 or 8 weeks old). However, the activity significantly increased after adolescence (middle age), thereafter decreasing with old age (over 100 weeks old) to a level equivalent to that found in young mice. In an additional strain difference study, O6-MT activities in liver extracts from 4 strains of mice were compared at 5 and 30 weeks of age. Although a similar age-associated increase of enzyme activity in adolescence was confirmed in all 4 strains investigated, the closed-colony ICR mice differed from the inbred strains in demonstrating significantly higher levels of O6-MT activity in females than in males. However, the same tendency was also observed in a comparison of the sexes in 30-week-old C3H/HeN, C57BL/6 and BALB/c mice.     

Osteoclast inhibitory lectin, an immune cell product that is required for normal bone physiology in vivo

Osteoclast inhibitory lectin (OCIL or clrb) is a member of the natural killer cell C-type lectins that have a described role mostly in autoimmune cell function. OCIL was originally identified as an osteoblast-derived inhibitor of osteoclast formation in vitro. To determine the physiological function(s) of OCIL, we generated ocil(-/-) mice. These mice appeared healthy and were fertile, with no apparent immune function defect, and phenotypic abnormalities were limited to bone. Histomorphometric analysis revealed a significantly lower tibial trabecular bone volume and trabecular number in the 10- and 16-week-old male ocil(-/-) mice compared with wild type mice. Furthermore, ocil(-/-) mice showed reduced bone formation rate in the 10-week-old females and 16-week-old males while Static markers of bone formation showed no significant changes in male or female ocil(-/-) mice. Examination of bone resorption markers in the long bones of ocil(-/-) mice indicated a transient increase in osteoclast number per unit bone perimeter. Enhanced osteoclast formation was also observed when either bone marrow or splenic cultures were generated in vitro from ocil(-/-) mice relative to wild type control cultures. Loss of ocil therefore resulted in osteopenia in adult mice primarily as a result of increased osteoclast formation and/or decreased bone formation. The enhanced osteoclastic activity led to elevated serum calcium levels, which resulted in the suppression of circulating parathyroid hormone in 10-week-old ocil(-/-) mice compared with wild type control mice. Collectively, our data suggest that OCIL is a physiological negative regulator of bone.     

Observations on soft tissue calcification in DBA/2NCrj mice in comparison with CRJ:CD-1 mice

Male and female DBA/2NCrj (DBA/2) mice 3, 4, 5 and 10 weeks old were examined biochemically and pathologically and the results obtained were compared with those for CRJ:CD-1 (ICR) mice of the same age. The plasma levels of glucose, triglyceride and total cholesterol tended to be lower in DBA/2 mice than in ICR mice but the levels of non-esterified fatty acid, calcium and inorganic phosphorus were almost the same in the two strains. The mean body weight of DBA/2 mice was significantly lower than that of ICR mice at each examination, and the relative weights of the hearts of male and female DBA/2 mice were significantly greater than those of male and female ICR mice. Cardiac calcinosis, tongue calcification and corneal degeneration occurred exclusively in DBA/2 mice with incidences of 30%-100%. The incidence and severity of these lesions increased with age but no sex differences were seen. It was difficult to relate differences in biochemical features of the two strains with pathological findings obtained in the DBA/2 mice. The numbers of cells secreting adrenocorticotropic hormone in the pituitary glands were significantly greater in male and female DBA/2 mice than in ICR mice, suggesting a higher secretion of corticosteroids in the former strain.     

Enterohepatic lesions in SCID mice infected with Helicobacter bilis

Helicobacter bilis is a recently identified species that colonizes the intestine and liver of mice. In immunocompetent mice, infections have been associated with mild hepatitis, and in immunocompromised mice, inflammatory bowel disease has been induced by intraperitoneal inoculation of the organism. We report inoculation of 6-week-old C.B-17 scid/scid mice by gastric gavage with approximately 10(7) H. bilis colony-forming units. Groups of mice were euthanized and necropsied 12, 24, and 36 weeks after inoculation. Mild to moderate proliferative typhlitis was evident in all mice at 12 and 36 weeks after inoculation and in most mice 24 weeks after inoculation. Mild to severe chronic active hepatitis was detected in 10 of 10 male mice and 3 of 10 female mice. These results indicate that H. bilis can cause moderate to severe enterohepatic disease in immunocompromised mice.     

Comparison of methods to diagnose an epizootic of Corynebacterium kutscheri pneumonia in rats

An outbreak of Corynebacterium kutscheri pneumonia occurred in a colony of laboratory rats. Diagnostic methods compared on a prospective basis included (1) an enzyme-linked immunosorbent assay (ELISA) for serum antibody to C. kutscheri; (2) C. kutscheri isolation from retrograde nasal wash, cervical lymph nodes, tracheal wash, lung homogenate; and (3) histopathology. C. kutscheri was isolated from one or more of the cultured sites from suspected cases, but no individual rat yielded C. kutscheri from all of the sites. The presence of histological lung lesions was the most frequent indicator of infection (8 of 9 suspected cases), followed by isolation of C. kutscheri from lung homogenate (5 of 6), ELISA for serum antibody (6 of 8), and isolation of C. kutscheri from cervical lymph node (4 of 8). Isolation of C. kutscheri from nasal wash (1 of 9) was the least sensitive indicator of infection. ELISA antibody was not detected in rats which had normal lungs and did not harbor C. kutscheri. It was concluded that ELISA provides noninvasive means for detecting infection and cervical lymph node culture increase the potential for successful isolation of the agent in C. kutscheri infected rats.