Morphologic and histologic comparisons between in vivo and nuclear transfer derived porcine embryos.

Nuclear transfer (NT) is an inefficient but invaluable tool of the biotechnology industry. This study looked at abnormalities associated with peri-implantation NT porcine embryos. Four experimental groups were examined: nonpregnant animals, in vivo pregnant animals, NT recipients, and manipulation control embryos (MC). Embryos (Day 10, 12, or 14) were evaluated for embryonic disc diameter, gross morphology, nucleoli density, and mitotic figure index. Day 12 (P < or = 0.03) and Day 14 (P < or = 0.01) NT embryos had increased numbers of nucleoli, and Day 14 NT embryos had an increased (P < or = 0.03) mitotic index compared to in vivo and MC embryos. In vivo produced Day 14 embryos had increased (P < or = 0.01) disk diameters when compared to other embryos except for MC Day 14, which also showed increases (P < or = 0.01) in disk diameter except when compared to in vivo produced Day 12 and Day 14 embryos. In vivo produced Day 12 had greater (P < or = 0.03) disk diameters when compared to NT and MC embryos except for MC Day 14, and in vivo produced Day 14 embryos, which had a significantly increased (P < or = 0.01) disk diameter. In vivo produced Day 14 embryos were morphologically more advanced (P < or = 0.01) than Day 14 NT and MC counterparts. NT embryos develop at a slower rate than their in vivo produced counterparts. The increase in nucleoli and mitotic index of NT embryos suggest the cell cycle may be affected or the NT embryos are employing other means to compensate for slow development. The techniques used during NT also appear to compromise embryo development.

Uterine responsiveness to estradiol and DNA methylation are altered by fetal exposure to diethylstilbestrol and methoxychlor in CD-1 mice: effects of low versus high doses.

We examined the effects on female CD-1 mice of fetal exposure to low doses of the drug diethylstilbestrol (DES) (0.1 microg/kg/day) and the insecticide methoxychlor (MXC) (10 microg/kg/day) as well as 1000-fold higher doses: 100 microg/kg/day DES and 10,000 microg/kg/day MXC. Pregnant females were administered these chemicals on gestation days 12-18. At 7-8 months of age, female offspring were ovariectomized and implanted for 7 days with a Silastic capsule containing estradiol. Relative to controls, females exposed to the 0.1 microg DES dose showed significantly heavier uteri, while females exposed to the 100 microg DES dose showed significantly lighter uteri. Females exposed prenatally to the 10 microg/kg dose of MXC had significantly heavier uteri relative to females exposed to the 10,000 microg/kg dose of MXC, but neither group differed significantly from controls. Liver weight for females exposed to both doses of DES was significantly greater than controls. Using a microarray approach to analyze DNA methylation, an increase in ribosomal DNA (rDNA) methylation was observed. Sequence data and Southern analysis indicate an increase in 18S rDNA and 45S pre-rDNA methylation in uterine samples exposed prenatally to low and high doses of DES. We thus found opposite effects of fetal exposure to a low and a high dose of DES on the uterine response to estradiol (inverted-U dose-response relationship). In contrast, there was a monotonic dose-response relationship found for prenatal DES exposure on both liver weight and ribosomal DNA hypermethylation.

Hamster polyomavirus infection in a pet Syrian hamster (Mesocricetus auratus).

An approximately 8-week-old pet Syrian hamster (Mesocricetus auratus) with a 1-week history of dyspnea, hyporexia, and ataxia was submitted for necropsy. On gross examination, the hamster had multiple abdominal adhesions and enlargement of the mesenteric lymph node. Histologic evaluation revealed multicentric lymphoma of the liver, jejunum, mesenteric lymph node, testicular fat pad, and epididymis. Based on the hamster's age and the type and distribution of the lymphoma, a presumptive diagnosis of hamster polyomavirus-induced lymphoma was made. A specific polymerase chain reaction (PCR) was developed, which confirmed the diagnosis. An in situ PCR demonstrated hamster polyomavirus DNA within lymphocytes of the multicentric lymphoma and renal tubular epithelial cells and within clusters of enterocytes in the jejunum. These data are consistent with environmental dissemination of hamster polyomavirus virions through the renal tubular epithelium and into the urine and with fecal shedding of hamster polyomavirus virions; however, additional studies will be needed to confirm these observations.

Differential interleukin-10 and gamma interferon mRNA expression in lungs of cilium-associated respiratory bacillus-infected mice.

The cilium-associated respiratory (CAR) bacillus is a gram-negative, extracellular bacterium that causes persistent respiratory tract infections in rodents. We have previously demonstrated that BALB/c mice are more susceptible to CAR bacillus-induced disease than resistant C57BL/6 mice, with elevations in pulmonary gamma interferon (IFN-gamma) and interleukin (IL)-4. IL-10 is a type 2 cytokine that can increase host susceptibility to bacterial diseases through its anti-inflammatory effects, including suppression of macrophage function. The purpose of this study was to further describe the cytokine profiles associated with histologic lesions in CAR bacillus-infected mice and to assess the effects of cytokine depletion on the pathogenesis of disease. Six-week-old female BALB/c and C57BL/6 mice and mice with targeted mutations in IFN-gamma and IL-4 were inoculated intratracheally with 10(5) CAR bacillus organisms, and samples were collected at 6 to 7 weeks postinoculation. Lung samples were collected for histopathologic examination and analysis of cytokine mRNA. IFN-gamma, IL-10, and IL-4 mRNA levels in the lungs of infected mice were semiquantitatively measured using a reverse transcriptase-mediated PCR assay and compared to those in uninfected control animals of each strain. BALB/c mice infected with CAR bacillus had a median lung lesion score of 6 and IL-10 and IL-4 mRNA levels were significantly elevated. The majority of C57BL/6 mice were resistant to disease characterized by lung lesions scores of 2 or less and a dominant IFN-gamma mRNA cytokine profile. A few C57BL/6 mice with lesions scores of 5 or greater had elevations in all three cytokines and were susceptible to disease. C57BL/6 IFN-gamma knockout mice had increased disease with elevations in IL-10 and IL-4 mRNA, while BALB/c IL-4 knockout mice infected with CAR bacillus had a mild decrease in lesion severity and an attenuated IL-10 mRNA expression compared to wild-type BALB/c mice. These data indicate that IL-10 and IL-4 predominate in CAR bacillus-induced histologic lesions in mice, while IFN-gamma may play a role in resistance to disease.

Diagnostic polymerase chain reaction assays for identification of murine polyomaviruses in biological samples.

PURPOSE: Mouse polyoma virus and K virus are murine polyomaviruses frequently used in carcinogenicity and cellular biology studies in mice. These viruses can cause persistent infections, which increase the likelihood of transmission through transplantation of cells from infected mice. To identify polyomavirus-infected biological samples, several diagnostic polymerase chain reaction (PCR) assays were developed. METHODS: Polyomavirus-family and virus-specific PCR assays were designed and optimized for specificity and sensitivity. The generic (polyomavirus-family) PCR assay and mouse polyoma virus-specific assays were compared with the mouse bioassay for diagnosis of infected cellular samples. RESULTS: Specificity of the PCR assays was confirmed by testing a battery of other murine viruses. The mouse polyoma virus PCR test was the most sensitive assay, detecting as few as 2,000 copies of homologous virus. The K virus PCR assay was about eightfold less sensitive, and the generic PCR test was the least sensitive. Mouse polyoma virus and generic PCR assays amplified mouse polyoma virus in the inoculum and tissues from experimentally infected mice, and performed better than did the mouse bioassay. CONCLUSIONS: Results of this study confirm that PCR is a specific and sensitive method for detection of murine polyomaviruses in biological samples.

Coxiella burnetii infection in C.B-17 scid-bg mice xenotransplanted with fetal bovine tissue.

Two from a group of approximately 50 C.B-17 scid-bg mice were examined because of lethargy, dehydration, and rough coat. Three months prior to development of clinical signs of disease, mice of this study had been surgically implanted with fetal bovine liver, thymus, and lymph node. At necropsy, marked splenomegaly and mild hepatomegaly were observed in both animals. Large areas of necrosis and inflammation, with associated intracytoplasmic granular basophilic inclusions, were observed in histologic sections of multiple organs. Aerobic and anaerobic culturing of the liver yielded negative results. Six months after the initial case, four more reconstituted scid-bg mice from a different fetal donor had identical clinical, gross, and histologic signs of disease. To determine whether the basophilic inclusions represented an infective agent, 4-month-old immune-naive C.B-17 scid-bg mice were inoculated intraperitoneally with a liver and spleen homogenate from an affected mouse. Two weeks after inoculation, mice developed clinical signs of disease and lesions identical to those seen in the signal mice. On ultrastructural examination of the liver, pleomorphic bacteria were found in large cytoplasmic vacuoles of hepatocytes. Bacterial DNA was amplified from the liver, using primers that amplify a segment of the 16S rRNA gene from many bacterial species. Sequencing of the polymerase chain reaction (PCR) product revealed gene sequence identical to that of Coxiella burnetii, the agent of Q-fever. These results highlight the need to consider infective agents of the donor species when working with xenografted animals.

Anatomic and physiologic reference values in least shrews (Cryptotis parva).

BACKGROUND AND PURPOSE: The least shrew is an established animal model for reproductive and pharmacologic research. Biologic reference data are needed to assess animal health status and provide a rationale for use of novel statistical programs to evaluate the effects of orally administered substances in toxicologic and pharmacologic studies. METHODS: Organ weights, blood biochemical and hematologic values, and food and water consumption data were collected from 50-day-old shrews after two weeks' consumption of a standard feline diet. RESULTS: In general, data correlated well with values reported for other mammalian species. Plasma phosphorus concentration was high. There was a significant difference in food and water consumption per gram of body weight between shrews at lower and upper (+/- 1 SD) weight ranges for the study. The 3.2-g animals consumed 27% more food per gram of body weight than did the 5.0-g animals. CONCLUSIONS: The high phosphorus concentration was attributed to hemolysis resulting from the axillary cut method of blood sample collection. The small size of the shrew allowed demonstration of the Kleiber effect within a +/- 1 SD weight range in a single species. The phenomenon necessitates the use of statistical methods other than the typical tests establishing the significance of the differences between the means of groups for oral toxicologic and pharmacologic studies.

Dietary genistein increased DMBA-induced mammary adenocarcinoma in wild-type, but not ER alpha KO, mice.

Dietary supplements containing concentrates of plant-derived estrogens are being increasingly used by consumers as alternatives for hormone replacement therapy, for treatment of menopausal symptoms, and as cancer preventives. The effect of dietary genistein on dimethylbenz[a]anthracene (DMBA)-induced mammary tumor development was investigated in wild-type (ER alpha WT) and estrogen receptor-alpha knockout (ER alpha KO) mice. ER alpha WT and ER alpha KO mice were fed a casein-based diet containing 0 or 1 g genistein/kg diet from weaning. Tumors were induced by oral administration of DMBA and subscapular implantation of medroxyprogesterone acetate. No tumors were observed in ER alpha KO mice. In ER alpha WT mice, dietary intake of genistein influenced tumor development, enhancing anaplasia of mammary cancer. Mice consuming genistein expressed malignant mammary adenocarcinoma, whereas benign adenomas were observed in mice fed the control diet. Dietary intake was also influenced by genistein, with ER alpha WT and ER alpha KO mice fed genistein consuming less food (p < 0.0001) and subsequently weighing less than mice fed the control diet (p < 0.0001). Significant differences in food intake by genotype were also observed (p = 0.0017), with ER alpha KO mice consuming less than ER alpha WT mice. Overall, this study found no protective effect of genistein on DMBA-induced mammary tumors in mice and suggests a potential adverse effect on tumor development when high levels of genistein are consumed.

Centrosome-centriole abnormalities are markers for abnormal cell divisions and cancer in the transgenic adenocarcinoma mouse prostate (TRAMP) model.

We utilized the transgenic adenocarcinoma mouse prostate (TRAMP) model to study the formation of abnormal mitosis in malignant tumors of the prostate. The results presented here are focused on centrosome and centriole abnormalities and the implications for abnormal cell divisions, genomic instability, and apoptosis. Centrosomes are microtubule organizing organelles which assemble bipolar spindles in normal cells but can organize mono-, tri-, and multipolar mitoses in tumor cells, as shown here with histology and electron microscopy in TRAMP neoplastic tissue. These abnormalities will cause unequal distribution of chromosomes and can initiate imbalanced cell cycles in which checkpoints for cell cycle control are lost. Neoplastic tissue of the TRAMP model is also characterized by numerous apoptotic cells. This may be the result of multipolar mitoses related to aberrant centrosome formations. Our results also reveal that centrosomes at the poles in mitotic cancer cells contain more than the regular perpendicular pair of centrioles which indicates abnormal distribution of centrioles during separation to the mitotic poles. Abnormalities in the centriole-centrosome complex are also seen during interphase where the complex is either closely associated with the nucleus or loosely dispersed in the cytoplasm. An increase in centriole numbers is observed during interphase, which may be the result of increased centriole duplication. Alternatively, these centrioles may be derived from basal bodies that have accumulated in the cell's cytoplasm, after the loss of cell borders. The supernumerary centrioles may participate in the formation of abnormal mitoses during cell division. These results demonstrate multiple abnormalities in the centrosome-centriole complex during prostate cancer that result in abnormal mitoses and may lead to increases in genomic instability and/or apoptosis.

Antibody and cytokine responses to the cilium-associated respiratory bacillus in BALB/c and C57BL/6 mice.

The cilium-associated respiratory (CAR) bacillus is a gram-negative, gliding bacterium that causes persistent respiratory tract infections in rodents despite histologic and serologic evidence of a marked immune response. To assess humoral immunity and cytokine responses in CAR bacillus disease, 6-week-old female BALB/c and C57BL/6 mice were inoculated intratracheally with 10(5) CAR bacillus organisms. CAR bacillus-specific serum immunoglobulins (immunoglobulin M [IgM], IgG1, IgG2a, IgG2b, IgG3, and IgA) and local pulmonary cytokines (tumor necrosis factor alpha [TNF-alpha], gamma interferon [IFN-gamma], and interleukin-4 [IL-4]) were evaluated by enzyme-linked immunosorbent assay every 7 days for 49 days. BALB/c mice developed CAR bacillus-induced lesions early in the course of disease that became more severe with time. Correlating with increasing disease severity, BALB/c mice had elevations in all antibody isotypes tested, and elevations in pulmonary TNF-alpha, IFN-gamma, and IL-4. C57BL/6 mice developed mild lesions with mild increases in serum IgM, IgG1, IgG2b, and IgG3 levels and minimally detectable IgG2a and IgA. Cytokine perturbations were not detected in C57BL/6 mice. The persistence of infection in BALB/c mice with vigorous serum antibody responses and increased IFN-gamma and IL-4 responses suggests that humoral immunity and T-cell responses are ineffective at preventing CAR bacillus disease. Furthermore, the lackluster antibody responses and undetectable cytokine responses in C57BL/6 mice suggest that humoral immunity and T-cell responses are not critical in resistance to CAR bacillus-induced disease.

Helicobacter mesocricetorum sp. nov., A novel Helicobacter isolated from the feces of Syrian hamsters.

A spiral-shaped bacterium with bipolar, single, nonsheathed flagella was isolated from the feces of Syrian hamsters. The bacterium grew as a thin spreading film at 37 degrees C under microaerobic conditions, did not hydrolyze urea, was positive for catalase and alkaline phosphatase, reduced nitrate to nitrite, did not hydrolyze hippurate, and was sensitive to nalidixic acid but resistant to cephalothin. Sequence analysis of the 16S rRNA gene and biochemical and phenotypic criteria indicate that the novel bacterium is a helicobacter. The novel bacterium is most closely related to the recently described mouse enteric helicobacter, Helicobacter rodentium. This is the first urease-negative Helicobacter species with nonsheathed flagella isolated from feces of asymptomatic Syrian hamsters. We propose to name this novel helicobacter Helicobacter mesocricetorum. The type strain is MU 97-1514 (GenBank accession number AF072471).

Enteric lesions in SCID mice infected with "Helicobacter typhlonicus," a novel urease-negative Helicobacter species.

BACKGROUND AND PURPOSE: Several rodent helicobacters have been associated with chronic active hepatitis or inflammatory bowel disease. Severe combined immunodeficient (SCID) mice appear to be inherently susceptible to disease attributable to these emerging pathogens. With the advent of polymerase chain reaction (PCR) analysis, it has become clear that several as yet unidentified Helicobacter species may also colonize rodents, but their capacity to cause disease is unknown. METHODS: A Helicobacter species isolated from feces of a BALB/c mouse and provisionally named "H. typhlonicus" was used to inoculate helicobacter-free 4-week-old SCID mice (n = 11 males and 11 females). At various weeks after inoculation, mice were sacrificed and liver and intestinal specimens were collected for histologic examination and PCR analyses. RESULTS: The C.B-17 scid/scid mice inoculated with "H. typhlonicus" developed moderate to severe proliferative typhlocolitis, similar to that seen in SCID mice infected with H. hepaticus or H. bilis. However, in contrast to mice infected with H. hepaticus or H. bilis, lesions of chronic active hepatitis were not detected in mice inoculated with "H. typhlonicus." A similar disease syndrome developed in SCID mice cohabitated with B6D2F1 mice naturally infected with a novel Helicobacter species that was genetically identical to "H. typhlonicus." CONCLUSION: "Helicobacter typhlonicus" joins a growing list of helicobacters that are capable of inducing enteric disease in immunodeficient mice.

Cloning and expression of an immunogenic membrane-associated protein of Helicobacter hepaticus for use in an enzyme-linked immunosorbent assay.

Helicobacter hepaticus is a bacterial pathogen that causes chronic active hepatitis and inflammatory bowel disease in mice. The purpose of this study was to develop a recombinant antigen-based enzyme-linked immunosorbent assay (ELISA) to detect H. hepaticus-infected mice. A genomic library of H. hepaticus was constructed and was screened with sera from H. hepaticus-infected mice. A 459-bp open reading frame that coded for an 18-kDa immunoreactive protein, MAP18, was identified. The gene had high identity with genes coding for outer membrane proteins of other bacteria, and the predicted amino acid sequence of MAP18 had a putative membrane-trafficking signal sequence and a putative signal peptidase II cleavage site. The recombinant protein was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein, GST-MAP18, and purified by affinity chromatography. The 44-kDa fusion protein was detected on Western blots probed with sera from H. hepaticus-infected mice but was not detected on blots probed with sera from mice infected with Helicobacter muridarum or Helicobacter bilis or with sera from mice free of Helicobacter infection. The GST-MAP18 fusion protein was used as an antigen in an ELISA to detect anti-H. hepaticus antibodies in sera from infected mice. This ELISA was compared to an H. hepaticus-specific ELISA that uses a detergent extract of H. hepaticus as the antigen. Sera from mice naturally and experimentally infected with H. hepaticus, H. bilis, or H. muridarum and sera from mice free of Helicobacter infection were evaluated. Both ELISAs performed with a high specificity (98%); however, the detergent extract-based ELISA performed with a higher sensitivity (89%) than the recombinant protein-based ELISA (sensitivity, 66%). These data indicate that H. hepaticus carries a gene that encodes an immunogenic 18-kDa membrane-associated protein; however, antibodies to this protein are not detected in all infected mice.

Interleukin-12 has a role in mediating resistance of murine strains to Tyzzer's disease.

Clostridium piliforme induces enterohepatic disease in many domestic and laboratory animal species. Susceptibility to infection is known to vary with the immune status and strain of the host, but little is known about specific immune mechanisms that regulate this disease. To evaluate host control of C. piliforme infection, we examined the role of interleukin-12 (IL-12) both in the control of and in the response to murine C. piliforme infection. For this study, 3-week-old C. piliforme-resistant C57BL/6 or -susceptible DBA/2 mice were infected intravenously with either the toxic H1 or the nontoxic M1 C. piliforme isolate. Serum and liver samples were collected prior to C. piliforme inoculation (day 0) and at days 1, 3, 7, 14, and 28 postinoculation. Evaluation of hepatic IL-12 p40 mRNA expression by reverse transcription-PCR and of total-IL-12 protein levels in serum by enzyme-linked immunosorbent assay revealed that C. piliforme induced elevations in both hepatic p40 mRNA and serum total-IL-12 levels at all times postinoculation. Elevations were similar with both toxic and nontoxic C. piliforme isolates. Levels of total IL-12 in serum were significantly (P < 0.05) higher in C57BL/6 mice than in DBA/2 mice. Additional experiments were performed in which polyclonal antibody treatment was used to neutralize IL-12 in mice of both strains prior to intravenous inoculation with toxic C. piliforme H1. IL-12 neutralization increased the severity of Tyzzer's disease at day 3 postinoculation in both mouse strains, but the degree of increase was greater in C57BL/6 mice than in DBA/2 mice.

Antigenic analyses of cilia-associated respiratory (CAR) bacillus isolates by use of monoclonal antibodies.

Mouse monoclonal antibodies (MAbs) developed to a rat isolate (R-3) of cilia-associated respiratory (CAR) bacillus were used to assess antigenic relationships among three rat and five rabbit CAR bacillus isolates. Evaluation of MAbs by enzyme-linked immunosorbent assays (ELISAs) indicated that 87 of 241 hybridomas secreted CAR bacillus-reactive antibodies that could be grouped into four major groups. Group-I MAbs reacted with epitopes expressed by all CAR bacillus isolates and at least two or more nonrelated species of bacteria. Group-II, -III, and -IV MAbs reacted with only one or more of the rat CAR bacillus isolates; no MAbs reacted only with rat and rabbit CAR bacillus isolates. Western blot analyses indicated that 41-, 50-, and 105-kDa peptides of rat CAR bacillus isolates expressed rat CAR bacillus group- and isolate-specific epitopes. Hyperimmune anti-CAR bacillus antiserum and serum specimens from a CAR bacillus histologically positive mouse and rat also reacted with the 41-, 50-, and 105-kDa peptides. Sera from CAR bacillus histologically negative rats did not react with these peptides. These results suggest that the 41-, 50-, and 105-kDa peptides may represent suitable antigens for development of a specific ELISA for detection of rodent CAR bacillus infections. Furthermore, these data indicate that use of crude CAR bacillus preparations for either rat or rabbit CAR bacillus ELISAs is inappropriate.

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.