Efficient differentiation of human neutrophils with recapitulation of emergency granulopoiesis in human G-CSF knockin humanized mice.

Neutrophils are critical mediators during the early stages of innate inflammation in response to bacterial or fungal infections. A human hematopoietic system reconstituted in humanized mice aids in the study of human hematology and immunology. However, the poor development of human neutrophils is a well-known limitation of humanized mice. Here, we generate a human granulocyte colony-stimulating factor (hG-CSF) knockin (KI) NOD/Shi-scid-IL2rgnull (NOG) mouse in which hG-CSF is systemically expressed while the mouse G-CSF receptor is disrupted. These mice generate high numbers of mature human neutrophils, which can be readily mobilized into the periphery, compared with conventional NOG mice. Moreover, these neutrophils exhibit infection-mediated emergency granulopoiesis and are capable of efficient phagocytosis and reactive oxygen species production. Thus, hG-CSF KI mice provide a useful model for studying the development of human neutrophils, emergency granulopoiesis, and a potential therapeutic model for sepsis.

Interstitial pneumonia in immunocompetent laboratory rats caused by natural infection with Pneumocystis carinii.

Pneumocystis (P.) carinii is known to cause fatal pneumonia in immunocompromised rats. Cases of P. carinii interstitial pneumonia in immunocompetent rats have been shown histologically to present with perivascular lymphoid cuffs, which have previously been attributed to rat respiratory virus. This study aims to determine the prevalence and pathological characteristics of P. carinii in immunocompetent laboratory rats in experimental facilities in Japan. An epidemiological survey for this agent was performed using PCR to assess 1,981 immunocompetent rats from 594 facilities in Japan. We observed that 6 of the 1,981 rats (0.30%) from 4 out of 594 facilities (0.67%) were positive for P. carinii without infection of other known pathogens. Gross pulmonary lesions were found in 4 of the 6 affected rats. The lungs of these rats contained scattered dark red/gray foci. Histopathologically, the lungs exhibited interstitial pneumonia with lymphoid perivascular cuffs: Pneumocystis cysts were observed using Grocott's methenamine silver stain. To our knowledge, this report is the first to reveal the prevalence of natural P. carinii infection in immunocompetent laboratory rats in Japan.

Pathogenesis of murine astrovirus in experimentally infected mice.

Astroviruses are often associated with gastrointestinal diseases in mammals and birds. Murine astrovirus (MuAstV) is frequently detected in laboratory mice. Previous studies on MuAstV in mice did not report any symptoms or lesions. However, little information is available regarding its pathogenicity in immunodeficient mice. Therefore, in this study, we experimentally infected germ-free NOD.Cg-PrkdcscidIl2rgtm1Sug/ShiJic (NOG) mice, which are severely immunodeficient, with MuAstV. Germ-free mice were used for experimental infection to eliminate the effects of intestinal bacteria. Mice in each group were then necropsied and subjected to PCR for MuAstV detection, MuAstV RNA quantification in each organ, and histopathological examination at 4 and 28 days post inoculation (DPI). Tissue samples from the small intestine were examined by transmission electron microscopy. No symptoms or abnormalities were detected in any mice during necropsy. The MuAstV concentration was highest in the lower small intestine, where it increased approximately 8-fold from 4 to 28 DPI. Transmission electron microscopy revealed circular virus particles of approximately 25 nm in diameter in the cytoplasm of the villous epithelial cells of the lower small intestine. Histopathological examination did not reveal any abnormalities, such as atrophy, in the intestinal villi. Our results suggest that MuAstV proliferates in the villous epithelial cells of the lower small intestine and has weak pathogenicity.

Prevalence of murine astrovirus in laboratory animal facilities in Japan.

To investigate the prevalence of murine astrovirus (MuAstV) in mice in laboratory animal facilities in Japan, a polymerase chain reaction (PCR) test targeting the RNA-dependent RNA polymerase (RdRP) gene was performed on the cecum contents of 1,212 mice (1,183 immunocompetent mice and 29 immunodeficient mice) from 226 facilities. The results showed that 118 (52.2%) of the 226 facilities were positive for MuAstV. Out of the 1,212 mice, 424 (35.0%) were positive. No gross lesions were observed in any of the mice examined. A phylogenetic analysis for 15 selected strains revealed that 13 strains formed one cluster, while two were genetically distant from that cluster. These results suggest that multiple strains are prevalent in laboratory mice in Japan.

Survey and Experimental Infection of Enteropathogenic Escherichia coli in Common Marmosets (Callithrix jacchus).

Common marmosets (Callithrix jacchus) are frequently used for biomedical research but can be afflicted with diarrhea-a serious and potentially lethal health problem. Enteropathogenic Escherichia coli (EPEC) is thought to be the causative pathogen of hemorrhagic typhlocolitis in common marmosets, but the actual incidence of the disease and the relationship between EPEC and hematochezia are unknown. This study investigated the prevalence of EPEC infection in common marmosets and the association between EPEC and hematochezia. A total of 230 stool or rectal swab samples were collected from 230 common marmosets (98 clinically healthy, 85 diarrhea, and 47 bloody stool samples) and tested by culture-based detection and PCR amplification of VT1, VT2, LT, ST, eae, and bfp genes. Healthy animals were divided into three groups (n = 4 each for high and low concentration groups and n = 2 as negative control), and those in the experimental groups were perorally inoculated with a 2-ml of suspension of EPEC R811 strain adjusted to 5 × 108 (high concentration) and 5 × 104 (low concentration) CFU/ ml. Two animals in each group were examined 3 and 14 days post-inoculation (DPI). EPEC was detected in 10 of 98 clinically healthy samples (10.2%), 17 of 85 diarrhea samples (20%), and all 47 bloody stool samples (100%), with a significant difference detected between presence of EPEC and sample status (P < 0.01). Acute hematochezia was observed in all animals of the high-concentration group but not in other groups at 1 or 2 DPI. A histopathological examination revealed the attachment of gram-negative bacilli to epithelial apical membranes and desquamated epithelial cells in the cecum of animals in the high-concentration group at 3 DPI. These findings suggest that EPEC is a causative agent of hemorrhagic typhlocolitis in common marmosets.

A case of nontraumatic gas gangrene in a common marmoset (Callithrix jacchus).

The common marmoset is widely used in neuroscience and regenerative medicine research. However, information concerning common marmoset disorders, particularly infectious diseases, is scarce. Here, we report a case of a female common marmoset that died suddenly due to gas gangrene. The animal presented with gaseous abdominal distention at postmortem, and Clostridium perfringens type A was isolated from several tissues. Vacuoles, a Gram-positive bacteremia and intravascular hemolysis were observed microscopically in the muscles, liver and lungs. On the basis of these findings, we diagnosed nontraumatic gas gangrene caused by Clostridium perfringens type A in this common marmoset.

Draft Genome Sequence of Enteropathogenic Escherichia coli, Isolated from the Bloody Stool Sample of a Common Marmoset (Callithrix jacchus).

Here, we report the draft genome sequence of Escherichia coli strain R811. This bacterium was isolated from the bloody stool sample of a common marmoset, and was categorized as enteropathogenic E. coli because it possessed eae.

Microbiological survey of mice (Mus musculus) purchased from commercial pet shops in Kanagawa and Tokyo, Japan.

Information regarding the prevalence of infectious agents in mice in pet shops in Japan is scarce. This information is particularly useful for minimizing the risk of potential transmission of infections to laboratory mice. Therefore, we surveyed infectious agents in mice from pet shops in Kanagawa and Tokyo, Japan. The survey was conducted in 28 mice from 5 pet shops to screen for 47 items (17 viruses, 22 bacteria and fungi, 10 parasites) using culture tests, serology, PCR, and microscopy. The most common viral agent detected was murine norovirus (17 mice; 60.7%), followed by Theiler's murine encephalomyelitis virus (13 mice; 46.4%), and mouse hepatitis virus (12 mice; 42.8%). The most common agent amongst the bacteria and fungi was Pasteurella pneumotropica (10 mice; 35.7%), followed by Helicobacter ganmani and Pneumocystis murina (8 mice; 28.5%, for both). Tritrichomonas muris was the most common parasite (19 mice; 67.8%), followed by Spironucleus muris (13 mice; 46.4%), Aspiculuris tetraptera, and Syphacia obvelata (8 mice each; 28.5%). Remarkably, a zoonotic agent, Hymenolepis nana, was found in 7 mice (25%). Given these results, we suggest that the workers in laboratory animal facilities should recognize again the potential risks of mice outside of the laboratory animal facilities as an infectious source, and avoid keeping mice as pets or as feed for carnivorous reptiles as much as possible for risk management.

Current microbiological status of laboratory mice and rats in experimental facilities in Japan.

On the basis of our 2011 microbiological monitoring tests, we report here the current microbiological status of mice and rats housed in experimental facilities in Japan. We tested more than 14,000 mice, 6,000 serum samples, 500 fecal or cecal samples, and 200 lung samples from 3,549 mouse facilities within Japanese universities and institutes (U/I), pharmaceutical companies and contract research organizations (P/C). We also tested more than 1,500 rats, 1,600 serum samples, and 20 fecal or cecal samples from 772 U/I and P/C rat facilities. Bacterial cultures, serology, microscopy, PCR, and DNA analysis using DNA chips were performed. Staphylococcus aureus (18.8% in mouse facilities, 58.6% in rat facilities) was the most prevalent agent in both the mouse and rat facilities. The next most prevalent agents in the mouse facilities were murine norovirus (11.97%), intestinal protozoa (0.05-8.49%, from various species), Pasteurella pneumotropica (5.32%), and Helicobacter hepaticus (3.17%), while intestinal protozoa (0.74-6.84% from various species), Syphacia muris (6.20%), Pseudomonas aeruginosa (3.61%), and Pasteurella pneumotropica (3.05%) were the subsequent most prevalent agents in the rat facilities. These results suggest that the currently prevalent microbes in laboratory mice and rats in Japan are mainly opportunistic pathogens, intestinal protozoa, and microbes with low pathogenicity.

Prevalence of Bordetella hinzii in mice in experimental facilities in Japan.

To reveal the current status of the prevalence of Bordetella hinzii in mice in experimental facilities in Japan, a survey of this agent was performed by culture of tracheal swabs from a total of 12,923 mice from 1699 facilities (12,192 mice from 1572 facilities in universities and research institutes and 731 mice from 127 facilities in pharmaceutical companies) in total. In the results, 195 out of 12,192 mice (1.6%) from 44 out of 1572 facilities (2.8%) in universities and research institutes were positive for B. hinzii. No B. hinzii-positive mice were found in 127 pharmaceutical companies surveyed. Gross lesions in the lungs with isolation of B. hinzii were observed in seven mice from four universities, and the lesions were identified as bronchopneumonia histopathologically. To our knowledge, this is the first report to reveal the prevalence of B. hinzii in laboratory mice.