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.     

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.     

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.     

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.     

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.     

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.     

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.     

Clostridium difficile typhlitis associated with cecal mucosal hyperplasia in Syrian hamsters.

A sudden increase in mortality occurred in a closed breeding colony of Syrian hamsters (Mesocricetus auratus). The colony consisted of approximately 40 hamsters, 8 of which were affected. Four adult males died suddenly. One pregnant female and one weanling died after having been observed as depressed for 1 day and 2 weeks respectively. One weanling and one adult male were euthanized. All affected hamsters had signs of diarrhea. At necropsy, hemorrhagic fluid-filled ceca were noted in five of eight animals. Clostridium difficile cytotoxin B was present in high titers [10(-3) to 10(-8)] in cecal contents of six of six animals tested, whereas C. difficile culture yielded positive results in only one of six animals. Histopathologically, findings consistent with Clostridium-induced typhlitis including necrosis, epithelial denudation, vascular congestion, and hemorrhage were present in six of six ceca evaluated. In addition, signs of a more chronic disease process included cecal mucosal hyperplasia in five of six hamsters. A silver stain of cecal hyperplastic mucosa for intracellular organisms including Campylobacter-like organisms was negative in all affected hamsters. Antibiotics had not been administrated to any hamster in this colony, nor had the affected animals been experimentally manipulated. Testing for antibiotic residues in the feed was negative, and C. difficile was not isolated from feed, water, or feces of unaffected hamsters. Thus C. difficile-induced typhlitis should be included in the differential diagnosis of deaths in hamsters which have no clinical histories of prior antibiotic administration or experimental manipulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characteristics of natural killer cells (NK cells) and features of the cytotoxic test in Syrian hamsters

The cytotoxic test in vitro with the use of xenogeneic target cells of human myeloma, strain K-562, labeled with 51Cr has demonstrated natural cytotoxicity of lymphoid cells from noninbred Syrian hamsters. This cytotoxicity occurs at the cost of non-adherent splenocytes. NK may be isolated over the gradient density of ficoll (1.078), selective for large granular lymphocytes. To detect the maximal lytic activity of NK from Syrian hamsters in the cytotoxic test in vitro, they should be brought into 10-12 hour contact with sensitive target cells K-562. In Syrian hamsters, the highest natural cytotoxicity is shown by the cells of the blood and spleen. In the bone marrow and thymus, it is little pronounced and is virtually absent from the peripheral lymph nodes.     

Characterization of a Haemophilus sp. isolated from a rabbit with conjunctivitis

A case of conjunctivitis in a rabbit caused by a Haemophilus sp. is described. This was the only organism that was isolated in large numbers from the infected eye. The uninfected eye of the same rabbit contained Staphylococcus epidermidis, Corynebacterium kutscheri, Proteus mirabilis and a Neisseria sp. Effects of infection with live Haemophilus sp. isolated from the subject rabbit was studied in the eyes of rabbits and mice. Swabbing live organisms in the normal rabbit eye produced blepharitis within 48 hours. Signs of blepharitis started to disappear after 10 days without treatment. No reaction was noted in the control eye (swabbed with sterile saline) of the rabbit. Swabbing live organisms or sterile saline in the normal mouse eye failed to produce visible lesions up to 4 weeks. Evidence suggests that the Haemophilus sp. was pathogenic for rabbits but not for mice.     

Susceptibility of Germ-free Mice to Infectious Megaenteron

Germ-free (GF)-ICR mice were shown to be less susceptible to oral inoculation with a pathogenic strain of Escherichia coli (E. coli 0115a, c: K(B)) than GF-CF#1 mice. In GF-CF#1 mice a large number of organisms were recovered from the intestinal wall from the cecum to the rectum 3 to 7 days after inoculation. Unlike those in GF-CF#1 mice, lesions in GF-ICR mice were localized in a part of the cecum and organisms were recovered only from the cecal wall and rarely from organs other than those of the alimentary tract. In both strains of mice, however, organisms were recovered in large number from the intestinal contents. Histopathology and immunofluorescence revealed organisms closely attached to the surface of the cecum, colon and rectal epithelia in GF-CF#1 mice but only in a part of the cecal epithelium in GF-ICR mice. After being in contact with conventional CF#1 mice for 21 days and then inoculated orally with the pathogenic E. coli, ex-GF-CF#1 mice died within 14 days with severe intestinal lesions, but ex-GF-ICR mice survived without lesions.     

Upregulation of galectin-3 by Corynebacterium kutscheri infection in the rat lung.

Corynebacterium (C) kutscheri and Staphylococcus aureus were isolated from two Sprague-Dawley (SD) rats with a hemisected spinal cord. Grossly, gray-white bulging foci and abscesses were distributed throughout the parenchyma of the lung. Pathologically, severe necrotizing lobar pneumonia with abscesses and fibrinous pleuritis were observed. Immunohistochemical analysis found accumulation of galectin-3 in alveolar macrophages and the alveolar interstitial region. No other viral or bacterial pathogens were detected in these animals. In addition, similar pathogenic changes and accumulation of galectin-3 were observed in the lungs of SD rats experimentally infected with C. kutscheri. Using northern blot analysis, the relative galectin-3 and GAPDH mRNA levels were 4.6 to 9.3 times higher in C. kutscheri-infected lung than in uninfected controls. These results demonstrate that a single C. kutscheri infection can induce the upregulation of galectin-3 in the lung and that this molecule may have an important pathogenic role in C. kutscheri infections in rats.     

Aerobic Oral Bacteria in Healthy Captive Snakes

Cotton swab samples were taken from the ventral surface of the mouth and from the proximal esophagus from 23 captive nonpoisonous snakes. The samples were cultured and investigated for aerobic bacteria. Both the mouth and the esophagus) samples of 6 snakes were negative. When the bacterial isolates of the mouth and the esophagus of the whole snake population were compared, it was found that the flora isolated from both locations were similar. However, when the samples of individual snakes were compared it was found that the same isolates were seldom found in both the mouth and the esophagus. The most common bacteria found were Pseudomonas sp., Alcaligenes-like organisms, Gram-positive rods and Gram-positive cocci belonging to the family Micrococcaceae. Important pathogens were seldom isolated. Salmonella virchow could be found from 2 snakes. The presence of bacteriologically negative samples, great variations in the composition of the flora between individual snakes, and the occurrence of typical environmental bacteria in the oral cavity all suggest that snakes lack a specific autochtonous flora: and the bacteria isolated from the oral cavity may be occasional environmental bacteria. The source of pathogens may be the environment, too.     

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.     

Characterization of some previously unclassified Pasteurellaceae isolated from hamsters

Bacteria isolated from purulent processes on the jaws of European hamsters (Cricetus cricetus) and from intestinal inflammatory processes in Syrian hamsters (Mesocricetus auratus), bred as laboratory animals have been shown to be phenotypically similar but not identical with Pasteurella pneumotropica. Deoxyribonucleic acid (DNA)-DNA hybridization studies indicate that with one exception, the strains represent two new species of the family Pasteurellaceae. In the absence of a close genomic relatedness to members of the genera Actinobacillus or Pasteurella or allied organisms, however, the two new taxa are described without any formal designation. The one exception was identified as Actinobacillus capsulatus, a species not previously isolated from hamsters.     

Characterization of some previously unclassified Pasteurellaceae isolated from hamsters

Bacteria isolated from purulent processes on the jaws of European hamsters ( Cricetus cricetus ) and from intestinal inflammatory processes in Syrian hamsters ( Mesocricetus auratus ), bred as laboratory animals have been shown to be phenotypically similar but not identical with Pasteurella pneumotropica . Deoxyribonucleic acid (DNA)–DNA hybridization studies indicate that with one exception, the strains represent two new species of the family Pasteurellaceae. In the absence of a close genomic relatedness to members of the genera Actinobacillus or Pasteurella or allied organisms, however, the two new taxa are described without any formal designation. The one exception was identified as Actinobacillus capsulatus , a species not previously isolated from hamsters.     

Occurrence of bacteria in the mouth from genera of Micrococcus,Kocuria, Nesterenkonia,Kytococcus and Dermacoccus

The aim of the study was to assess the prevalence of different bacteria in the oral cavity. The bacteria were present in the oral cavities of 73 (48.7%) of 150 individuals. Nesterenkonia halobia, the most frequently isolated species, was found in 20 (27%) individuals, Micrococcus luteus in 16 (22%), Kocuria kristinae in 12 (16%), Kocuria varians in 10 (14%), Dermacoccus sedentarius in 9 (12%), Micrococcus lylae in 8 (11%), and Kytococcus nishinomiyaensis in 3 (4%). Mean counts of these microorganisms were relatively low and amounted in log10 CFU/ml saliva for M. luteus 1.87 +/- 0.52, for M. lylae 2.03 +/- 0.39, for N. halobia 2.14 +/- 0.56, for K. kristinae 2.20 +/- 0.69, for K. varians 2.19 +/- 0.67, for K. nishinomiyaensis 1.72 +/- 0.39, and for D. sedentarius 2.27 +/- 0.55. The factor limiting the population sizes of these microorganisms was most probably the antagonistic activity of the bacteria living in oral cavity.     

Clostridium difficile infection in adult hamsters.

Diarrhea was encountered in a group of adult female golden Syrian hamsters (Mesocricetus auratus) used for titrating the scrapie agent. Ninety percent of the cases occurred in animals over 210 days old even though animals of all age groups lived in the colony concurrently. The cause of diarrhea was investigated in both uninoculated animals and those receiving greater than a limiting dilution of scrapie infectivity, i.e., animals that were not expected to contract the experimental scrapie disease. Three forms of diarrhea were observed. The most commonly encountered was profuse and watery. A chronic form presented with semiformed, thin fecal material smearing the retroperitoneal region. Hemorrhagic diarrhea was observed rarely. Mortality was high among animals with acute watery or hemorrhagic diarrhea. Animals with semiformed soft stools were dehydrated, had a roughened hair-coat, and hunched back. Cardinal lesions were necrosis, inflammation, and mucosal hyperplasia of the cecum and colon and cholangiohepatitis with amyloid deposition. Diffuse renal amyloidosis was present in chronic cases. Toxigenic, cytotoxin B-positive Clostridium difficile was isolated from a majority of affected animals. Cytotoxin B was also present in cecal homogenates of diarrheic animals with C. difficile. The pathological and microbiologic findings indicated a typhlitis and colitis in adult hamsters that was associated with C. difficile infection.     

Nitric oxide synthase in the enteric nervous system of the guinea-pig: a quantitative description

The distribution and abundance of nitric oxide synthase (NOS)-containing neurons and their terminals in the gastrointestinal tract of the guinea-pig were examined in detail using NADPH diaphorase histochemistry and NOS immunohistochemistry. NOS-containing cell bodies were found in the myenteric plexus throughout the gastrointestinal tract and in the submucous plexus of the stomach, colon and rectum. NOS-containing neurons comprised between 12% (in the duodenum) and 54% (in the esophagus) of total myenteric neurons. In the ileum, NOS neurons represented 19% of total myenteric neurons. Most of the NOS neurons throughout the gastrointestinal tract possessed lamellar dendrites and a single axon. NOS-containing terminals were abundant in the circular muscle, including that of the sphincters, but were rare in the longitudinal muscle, except for the taeniae of the caecum. The muscularis mucosae of the esophagus, stomach, colon and rectum received a medium to dense innervation by NOS terminals. Within myenteric ganglia, NOS-containing terminals were extremely sparse in the esophagus, stomach and duodenum, common in the ileum and distal colon and extremely dense in the proximal colon and rectum. The submucous plexus in the ileum and large intestine contained a sparse plexus of NOS-containing terminals. NOS terminals were not observed in the mucosa of any region. We conclude that throughout the gastrointestinal tract of the guinea-pig, NOS neurons are inhibitory motor neurons to the circular muscle; in the ileum and large intestine, NOS neurons may also function as interneurons.     

Transdiaphragmatic lymphatic transport of intraperitoneally administered marker in hamsters.

OBJECTIVE: To determine whether transdiaphragmatic transport in hamsters is similar to that described in other animals by examining transport of an intraperitoneally administered marker. METHODS: Monastral blue B suspension was administered intraperitoneally to 28 male Syrian hamsters (Mesocricetus auratus). Four hamsters each were euthanized 7, 15, and 30 min, and 1, 2, 3, and 24 h later. Specimens were examined microscopically for presence of marker. RESULTS: Marker was present in intrathoracic lymphatic vessels and cranial and caudal mediastinal lymph nodes by 7 min after its administration. The amount of marker in lymph nodes increased with time. The subcapsular distribution of marker was consistent with lymphatic transport. By 1 h after its administration, marker was present in the liver, spleen, bone marrow, and mesenteric and mandibular lymph nodes. Patterns of marker distribution in these tissues were consistent with hematogenous transport, but the amount of marker was considerably less than that in the intrathoracic lymph nodes at corresponding times. CONCLUSIONS: Particulates were most likely translocated from the hamster peritoneal cavity to intrathoracic lymph nodes via transdiaphragmatic lymphatic vessels. A portion of the translocated particulates entered the blood, where they were distributed to a variety of tissues within a short time.