Detection and Transmission of Proteus mirabilis in Immunodeficient Mice.

The exclusion of opportunistic pathogens is important for protecting animal health and ensuring desired research outcomes in highly immunodeficient mice. Proteus mirabilis has been associated with gastrointestinal tract lesions, septicemia, pyelonephritis, splenomegaly, and hepatitis and can influence select mouse models. To inform health-surveillance practices after we experienced difficulty in excluding P. mirabilis from our mouse colony, we aimed to determine the likelihood of detecting P. mirabilis-positive immunocompromised (SRG), immunovague (Fbn1+/-), and immunocompetent (CD1) colony mice through culture and PCR testing; to evaluate transmission via 2 sentinel-based approaches (direct contact and indirect dirty-bedding transfer); and to further characterize associated pathology. We hypothesized that immunocompromised mice would be better detectors and transmitters of P. mirabilis. Multiple logistic regression models were used for analysis and included PCR copy number, repeated testing, age, sex, and antibiotic-treated (trimethoprim-sulfamethoxazole) diet as covariates. Repeated testing over 10 wk showed that P. mirabilis -colonized immunocompromised colony mice were 95 times more likely than immunocompetent mice to test positive by culture and 30 times more likely by PCR assay. Sentinel mice were 15 times more likely to test positive by PCR assay for P. mirabilis when exposed by direct contact compared with dirty bedding and 18 times more likely to test positive when exposed to positive immunocompromised as compared with immunocompetent colony mice. After 10 wk of exposure, 3.8% of dirty-bedding sentinel PCR tests were positive, as compared with 30.7% of contact sentinels. Only immunocompromised mice on antibiotic diet (37.5%) developed lesions of the urogenital tract and abdominal cavity consistent with known pathology of P. mirabilis. Our findings suggest that PCR testing of dirty-bedding sentinels alone is not sufficient for the detection of P. mirabilis in mouse colonies. Direct-contact sentinels and testing of colony mice-especially if immunocompromised-with adjunct culture may facilitate successful bioexclusion.

Characterization of Multi-Drug Resistant Enterococcus faecalis Isolated from Cephalic Recording Chambers in Research Macaques (Macaca spp.).

Nonhuman primates are commonly used for cognitive neuroscience research and often surgically implanted with cephalic recording chambers for electrophysiological recording. Aerobic bacterial cultures from 25 macaques identified 72 bacterial isolates, including 15 Enterococcus faecalis isolates. The E. faecalis isolates displayed multi-drug resistant phenotypes, with resistance to ciprofloxacin, enrofloxacin, trimethoprim-sulfamethoxazole, tetracycline, chloramphenicol, bacitracin, and erythromycin, as well as high-level aminoglycoside resistance. Multi-locus sequence typing showed that most belonged to two E. faecalis sequence types (ST): ST 4 and ST 55. The genomes of three representative isolates were sequenced to identify genes encoding antimicrobial resistances and other traits. Antimicrobial resistance genes identified included aac(6')-aph(2"), aph(3')-III, str, ant(6)-Ia, tetM, tetS, tetL, ermB, bcrABR, cat, and dfrG, and polymorphisms in parC (S80I) and gyrA (S83I) were observed. These isolates also harbored virulence factors including the cytolysin toxin genes in ST 4 isolates, as well as multiple biofilm-associated genes (esp, agg, ace, SrtA, gelE, ebpABC), hyaluronidases (hylA, hylB), and other survival genes (ElrA, tpx). Crystal violet biofilm assays confirmed that ST 4 isolates produced more biofilm than ST 55 isolates. The abundance of antimicrobial resistance and virulence factor genes in the ST 4 isolates likely relates to the loss of CRISPR-cas. This macaque colony represents a unique model for studying E. faecalis infection associated with indwelling devices, and provides an opportunity to understand the basis of persistence of this pathogen in a healthcare setting.

Male Syrian Hamsters Experimentally Infected with Helicobacter spp. of the H. bilis Cluster Develop MALT-Associated Gastrointestinal Lymphomas.

BACKGROUND: Aged hamsters naturally infected with novel Helicobacter spp. classified in the H. bilis cluster develop hepatobiliary lesions and typhlocolitis. METHODS: To determine whether enterohepatic H. spp. contribute to disease, Helicobacter-free hamsters were experimentally infected with H. spp. after suppression of intestinal bacteria by tetracycline treatment of dams and pups. After antibiotic withdrawal, weanlings were gavaged with four H. bilis-like Helicobacter spp. isolated from hamsters or H. bilis ATCC 43879 isolated from human feces and compared to controls (n = 7 per group). RESULTS: Helicobacter bilis 43879-dosed hamsters were necropsied at 33 weeks postinfection (WPI) due to the lack of detectable infection by fecal PCR; at necropsy, 5 of 7 were weakly PCR positive but lacked intestinal lesions. The remaining hamsters were maintained for ~95 WPI; chronic H. spp. infection in hamsters (6/7) was confirmed by PCR, bacterial culture, fluorescent in situ hybridization, and ELISA. Hamsters had mild-to-moderate typhlitis, and three of the male H. spp.-infected hamsters developed small intestinal lymphoma, in contrast to one control. Of the three lymphomas in H. spp.-infected hamsters, one was a focal ileal mucosa-associated lymphoid tissue (MALT) B-cell lymphoma, while the other two were multicentric small intestinal large B-cell lymphomas involving both the MALT and extra-MALT mucosal sites with lymphoepithelial lesions. The lymphoma in the control hamster was a diffuse small intestinal lymphoma with a mixed population of T and B cells. CONCLUSIONS: Results suggest persistent H. spp. infection may augment risk for gastrointestinal MALT origin lymphomas. This model is consistent with H. pylori/heilmannii-associated MALT lymphoma in humans and could be further utilized to investigate the mechanisms of intestinal lymphoma development.

Spatial and temporal colonization dynamics of segmented filamentous bacteria is influenced by gender, age and experimental infection with Helicobacter hepaticus in Swiss Webster mice.

In this study, we examined colonization dynamics of segmented filamentous bacteria (SFB) in intestine of Swiss Webster (SW) mice infected with Helicobacter hepaticus (Hh). At 8 weeks post-inoculation with Hh (WPI), cecal and colonic SFB levels in the control males were significantly lower compared to those at 16 WPI. Hh infection in both genders did not alter SFB levels in the jejunum and ileum, but increased SFB levels in the cecum and colon of males compared to the controls (P < 0.05) at 8 WPI. At 16 WPI, the Hh-infected females contained lower levels of SFB in the jejunum, cecum and colon compared to the female controls. Irrespective of gender, aging and Hh infection, the Il-17A mRNA levels decreased from the small intestine to the cecum and then to the colon, whereas the Foxp3 mRNA levels were comparable in these intestinal regions. There were significant differences in Il-17A mRNA levels in the ileum (P < 0.05, R(2) = 0.31), with females having greater Il-17A mRNA levels than males, and higher SFB colonization levels related to more Il-17A mRNA. These results indicate that aging and gender play an important role in colonization dynamics of intestinal SFB and ileal SFB-associated Th17 response.

Impaired cholecystokinin-induced gallbladder emptying incriminated in spontaneous "black" pigment gallstone formation in germfree Swiss Webster mice.

"Black" pigment gallstones form in sterile gallbladder bile in the presence of excess bilirubin conjugates ("hyperbilirubinbilia") from ineffective erythropoiesis, hemolysis, or induced enterohepatic cycling (EHC) of unconjugated bilirubin. Impaired gallbladder motility is a less well-studied risk factor. We evaluated the spontaneous occurrence of gallstones in adult germfree (GF) and conventionally housed specific pathogen-free (SPF) Swiss Webster (SW) mice. GF SW mice were more likely to have gallstones than SPF SW mice, with 75% and 23% prevalence, respectively. In GF SW mice, gallstones were observed predominately in heavier, older females. Gallbladders of GF SW mice were markedly enlarged, contained sterile black gallstones composed of calcium bilirubinate and <1% cholesterol, and had low-grade inflammation, edema, and epithelial hyperplasia. Hemograms were normal, but serum cholesterol was elevated in GF compared with SPF SW mice, and serum glucose levels were positively related to increasing age. Aged GF and SPF SW mice had deficits in gallbladder smooth muscle activity. In response to cholecystokinin (CCK), gallbladders of fasted GF SW mice showed impaired emptying (females: 29%; males: 1% emptying), whereas SPF SW females and males emptied 89% and 53% of volume, respectively. Bilirubin secretion rates of GF SW mice were not greater than SPF SW mice, repudiating an induced EHC. Gallstones likely developed in GF SW mice because of gallbladder hypomotility, enabled by features of GF physiology, including decreased intestinal CCK concentration and delayed intestinal transit, as well as an apparent genetic predisposition of the SW stock. GF SW mice may provide a valuable model to study gallbladder stasis as a cause of black pigment gallstones.

Laser-assisted in vitro fertilization facilitates fertilization of vitrified-warmed C57BL/6 mouse oocytes with fresh and frozen-thawed spermatozoa, producing live pups.

The utility of cryopreserved mouse gametes for reproduction of transgenic mice depends on development of assisted reproductive technologies, including vitrification of unfertilized mouse oocytes. Due to hardening of the zona pellucida, spermatozoa are often unable to penetrate vitrified-warmed (V-W) oocytes. Laser-assisted in vitro fertilization (LAIVF) facilitates fertilization by allowing easier penetration of spermatozoa through a perforation in the zona. We investigated the efficiency of V-W C57BL/6NTac oocytes drilled by the XYClone laser, compared to fresh oocytes. By using DAP213 for cryoprotection, 83% (1,470/1,762) of vitrified oocytes were recovered after warming and 78% were viable. Four groups were evaluated for two-cell embryo and live offspring efficiency: 1) LAIVF using V-W oocytes, 2) LAIVF using fresh oocytes, 3) conventional IVF using V-W oocytes and 4) conventional IVF using fresh oocytes. First, the groups were tested using fresh C57BL/6NTac spermatozoa (74% motile, 15 million/ml). LAIVF markedly improved the two-cell embryo efficiency using both V-W (76%, 229/298) and fresh oocytes (69%, 135/197), compared to conventional IVF (7%, 12/182; 6%, 14/235, respectively). Then, frozen-thawed C57BL/6NTac spermatozoa (35% motile, 15 million/ml) were used and LAIVF was again found to enhance fertilization efficiency, with two-cell embryo rates of 87% (298/343) using V-W oocytes (P<0.05, compared to fresh spermatozoa), and 73% (195/266) using fresh oocytes. Conventional IVF with frozen-thawed spermatozoa using V-W (6%, 10/168) and fresh (5%, 15/323) oocytes produced few two-cell embryos. Although live offspring efficiency following embryo transfer was greater with conventional IVF (35%, 18/51; LAIVF: 6%, 50/784), advantage was seen with LAIVF in live offspring obtained from total oocytes (5%, 50/1,010; conventional IVF: 2%, 18/908). Our results demonstrated that zona-drilled V-W mouse oocytes can be used for IVF procedures using both fresh and frozen-thawed spermatozoa, producing live pups. The ability to cryopreserve mouse gametes for LAIVF may facilitate management of large-scale transgenic mouse production facilities.

Noninvasive temporal artery thermometry as an alternative to rectal thermometry in research macaques ( Macaca spp.).

Obtaining an animal's body temperature is essential for the assessment of its clinical status. For many species, rectal thermometry is the technique used most often; however, this method in macaques typically requires sedation or considerable physical restraint. A noninvasive and inexpensive temporal artery (TA) thermometer was evaluated as an alternative method for collecting body temperature measurements from macaques used in neuroscience research. Rectal and arterial temperatures were obtained from 86 macaques (mean age, 10.2 y) that had received ketamine (10 mg/kg IM) or Telazol (5 mg/kg IM); the arterial measurements were taken from behind the right ear. In addition, arterial temperatures were measured behind both ears in a cohort of awake, chaired macaques with cephalic restraint pedestals only (n = 8) or with cephalic restraint pedestals and recording chambers (n = 14). Within-subject repeatability for TA thermometry and agreement between rectal and arterial temperature measurements were assessed by using the Bland-Altman method. Temperature measurements indicated that values from TA thermometry were lower than those from rectal thermometry by 1.57 °C with a 95% agreement limit of ± 1.27 °C. Results show satisfactory repeatability with TA thermometry and agreement between arterial and rectal temperatures, demonstrating that TA thermometry can be a valuable tool in conscious, chaired macaques with restrained heads.