Xenopus laevis, commonly known as the South African Clawed frog, is a hardy adaptable species that is relatively easy to maintain as a laboratory animal. Gametogenesis in wild Xenopus laevis is continuous and under ideal conditions, reproduction can occur year round. This unique aspect of amphibian reproduction offers an advantage over mammalian model systems: the eggs and oocytes collected from laboratory maintained Xenopus laevis provide an abundant and readily obtainable supply of material for cellular and biological research. However, many investigators report that laboratory Xenopus laevis go through periods of unexplained inefficient or complete failure of oocyte production or the production of poor quality oocytes. This results in experimental delays, inability to reproduce data, and ultimately the use of more animals. There is a lack of evidenced based information regarding the housing conditions that are necessary to optimize the health and fecundity of this species in captivity, but studies of wild Xenopus laevis have shown that temperature, age of the female, and nutrition are of key importance. The objective of this report is to review oogenesis with a special emphasis on these factors as they pertain to laboratory Xenopus laevis maintained for the purpose of providing a steady supply of eggs and oocytes. Harvesting methods and other experimental techniques that affect the quality of eggs and oocytes are also discussed.

Background and Purpose: Mycoplasma haemomuris, a small pleomorphic bacterium parasitic of red blood cells, often causes chronic and subclinical infection of rodents. Mycoplasma haemomuris is uncultivable, and a serologic testing method is not readily available. The purpose of the study reported here was to develop a sensitive and specific polymerase chain reaction (PCR) test for detection of M. haemomuris in blood samples.Methods: On the basis of the regions of the M. haemomuris 16S rRNA gene most divergent from corresponding regions of related bacteria, M. haemomuris-specific primers were designed so that these primers could selectively amplify M. haemomuris DNA. A PCR test was performed, using blood samples from BALB/c mice infected with M. haemomuris strains TR 8564, TR 8563, and TR 8556.Results: Use of the PCR test enabled detection of M. haemomuris DNA in a minimum of 0.0001 μl of infected mouse blood. The test also was specific for M. haemomuris and did not amplify closely related species, such as M. haemofelis, M. haemosuis, M. orale, or Anaplasma marginale.Conclusion: This method is sensitive and specific for detection of M. haemomuris.

The purpose of the pilot study reported here was to evaluate serum and fecal total and allergen-specific immunoglobulin E (IgE) responses to dietary change in five Maltese x beagle dogs with suspected food hypersensitivity, compared with those of five clinically normal dogs. Clinical parameters (pruritus, otitis, and diarrhea) improved in the Maltese x beagle dogs during feeding of a novel diet, and signs were exacerbated by oral allergen provocation. Relative concentrations of serum and fecal wheat-, corn-, and milk-specific IgE were determined by use of an ELISA. The onset of clinical signs of disease was accompanied by an increase in serum allergen-specific IgE concentrations. In contrast, changes in clinical signs of disease or allergen-specific IgE values were not seen in the control group undergoing the same regimen. Total serum IgE concentration was measured by use of the ELISA, and comparison with known quantities of a monoclonal IgE allowed absolute values to be reported. Values were high in the Maltese x beagle colony (7 to 34 μg/ml), compared with those in the control dogs (0.7 to 6 μg/ml). Total serum and total fecal IgE concentrations did not change in either group during the study. Although allergen-specific IgE was detected in the feces of both groups, significant interassay variability made interpretation of the results difficult. The authors concluded that these Maltese x beagle dogs satisfied the currently recognized clinical criteria for the diagnosis of canine food hypersensitivity. Furthermore, the clinical and serologic responses seen in these dogs in response to oral allergen provocation suggest that this may be a useful model for the study of spontaneous food hypersensitivity.

Cilia-associated respiratory (CAR) bacillus is an unclassified, gram-negative, extracellular bacterium that causes chronic respiratory tract disease in rodents. Infected mice develop microscopic lesions characterized by a primary lymphocytic response followed by macrophage and neutrophilic infiltration. To characterize the lymphocytic subsets that respond to CAR bacillus infection, BALB/c mice were inoculated with 10 5 CAR bacillus bacteria. At seven weeks after inoculation, mice were euthanized and the tracheobronchiolar and hilar lymph nodes were collected and stained for cell surface markers to T cells (CD3, CD4, and CD8), B cells (B220, CD5), natural killer (NK) cells (pan-NK) and intracellular interleukin 10 (IL-10) and interferon-γ (IFN-γ). Flow cytometric analysis of lymph nodes from CAR bacillus-infected mice revealed 11% increase in frequency of B cells (B220+), 12% increase in the frequency of double-negative (CD4−CD8−CD3+) T cells, and slight increase in the B-1 subset of B cells (B220+CD5+). There was no change in the frequency of NK cells. The CAR bacillus-infected mice had an overall decrease in the frequency of T cells. Intracellular cytokine staining revealed distinct populations of T cells producing IL-10 and IFN-γ, and IL-10 production from B cells; NK cells were not a substantial source of IFN-γ. To our knowledge, this is the first characterization of lymphocytic responses and suggestion that B cells and double-negative T cells may be principally responsible for the lesions associated with CAR bacillus infection.

A variety of bone chambers are used in orthopedic research to study bone and tissue ingrowth in small and large animals. If different bone chambers are placed in one species, differences in bone ingrowth are observed. For instance, bone ingrowth in the bone conduction chamber (BCC) is high, but is low or absent in the repeated sampling bone chamber (RSBC).This difference may be explained by the design and fixation of these chambers. It is known that stress shielding and micromovement can influence bone formation. The objective of the study reported here was to determine whether stress shielding or soft tissue movement affected bone ingrowth in the BCC in the goat. Two types of caps were made, with fixation similar to that of the fixation plate of the RSBC. By placing the caps over the BCCs and fixating the caps directly to the tibial bone, the effect of stress shielding was studied. One cap was in direct contact with the bone chamber underneath, the other cap did not touch the chamber. This difference was used to observe whether movement of the soft tissue on top of the chamber and cap would affect bone ingrowth. Each limb received one control chamber without a cap and a chamber with a cap, either with or without contacting the BCC, yielding four implants per goat. After 12 weeks, bone and total tissue ingrowths were measured.Bone ingrowth was seen in 38 of 40 chambers. Total tissue and bone ingrowths were comparable between control chambers and BCCs with a cap, irrespective of type. Neither stress shielding, nor lack of movement of soft tissue affected bone ingrowth. Other factors in the design of the chambers were responsible for the difference in bone ingrowth between the BCC and the RSBC.

Successful immune reconstitution would enhance resistance of beige/scid mice to chronic infection with Mycobacterium avium subspecies paratuberculosis, but may cause damage to intestinal tissue. Therefore, we investigated the effect of adoptive transfer of BALB/c mouse splenocytes on lesion severity and intestinal physiology in beige/scid mice infected with M. paratuberculosis. Mice were inoculated intraperitoneally (i.p.) with M. paratuberculosis, and two weeks later were inoculated i.p. with viable spleen cells from immune-competent BALB/c mice. Mice were necropsied 12 weeks after infection when engraftment of lymphocytes, clinical disease, pathologic lesions, and intestinal electrophysiologic parameters were evaluated. Lymphocytes were rare in control beige/scid mice not inoculated with spleen cells. In contrast, high numbers of CD4+, CD8+, and B220+ lymphocytes were detected in the spleen of all beige/scid mice (n = 24) inoculated with spleen cells, indicating that adoptive transfer resulted in successful engraftment of donor lymphocytes (immune reconstitution). Immune reconstitution of M. paratuberculosis-infected beige/scid mice significantly reduced the severity of clinical disease and pathologic lesions, and numbers of bacteria in the liver. However, intestinal electrophysiologic parameters studied in vitro indicated that intestinal tissues from reconstituted beige/scid mice had reduced short-circuit current responses (due to reduced ion secretion) following electrical, glucose, and forskolin stimulation. These abnormal responses suggested that neural or epithelial cells in the intestine were damaged. We conclude that successful immune reconstitution of beige/scid mice enhance their resistance to M. paratuberculosis infection, but may cause pathophysiologic changes associated with intestinal inflammation.

The objective of the study reported here was to elucidate the optimal equilibration conditions for carrying out vitrification of two-cell mouse embryos, using a solution containing 2M dimethyl sulfoxide, 1M acetamide, and 3M propylene glycol (DAP213) as a cryoprotectant.Embryos were subjected to an equilibration process under 20 conditions of a combination of different temperatures (10 to 37°C) and times (5 to 90 sec), and viability of the embryos was assessed by the rate of development into blastocysts and into live fetuses. As a result, these rates of development into blastocysts did not differ from those for unfrozen embryos. The rate of development of frozen-thawed embryos into live fetuses under conditions of 30 sec. at 20°C, which was selected as having by highest operability, was 55.2%, comparable to the value (65.0%) for unfrozen embryos. Thus, the optimal equilibration condition for vitrification of two-cell mouse embryos, using DAP213 solution, was 30 sec at 20°C, under which embryo viability was maximized, and this equilibration process was considered useful as a practical two-cell embryo freezing process in the vitrification method.

Polymerase chain reaction (PCR) analysis is the standard method for detection of Helicobacter spp. infections in laboratory rodents, with H. hepaticus, H. bilis, and H. typhlonius considered primary pathogens. Fluorogenic nuclease PCR assays that detect all known rodent Helicobacter spp., or that specifically detect H. hepaticus, H. bilis, or H. typhlonius were developed to eliminate post-PCR processing, enhance specificity, and provide quantitative data on starting template concentration. Each fluorogenic PCR assay detected a minimum of 10 copies of target template, had comparable or greater sensitivity when compared directly with corollary gel detection PCR assays, and detected only targeted species when numerous Helicobacter spp. and other enteric bacteria were analyzed. Fluorogenic nuclease PCR analysis of fecal DNA samples obtained from numerous laboratory mice sources detected all samples with positive results by use of Helicobacter spp., H. hepaticus, H. bilis, and/or H. typhlonius gel detection PCR analysis, except for one sample that had positive results by H. typhlonius gel detection PCR but negative results by H. typhlonius fluorogenic nuclease PCR analysis. Among fecal DNA samples that were Helicobacter spp. negative by use of all gel detection PCR assays, the fluorogenic nuclease PCR assays detected target template in only one sample that was positive by use of the Helicobacter spp. and the H. bilis fluorogenic nuclease PCR assays. In conclusion, fluorogenic nuclease PCR assays provide sensitive, specific, and high-throughput diagnostic assays for detection of Helicobacter spp., H. hepaticus, H. bilis, and H. typhlonius in laboratory rodents, and the quantitative data generated by these assays make them potentially useful for bacterial load determination.

Infections with capillarid nematodes were observed in zebrafish (Danio rerio) kept at several research facilities and in a large carcinogen exposure study previously conducted at Oregon State University. We report a morphologic description that identifies the worm as Pseudocapillaria tomentosa, a common nematode of cyprinid and other fishes. Pathologic lesions associated with the infection ranged from inflammatory changes to aggressive neoplasms of the intestine (i.e., intestinal carcinomas and mixed malignant neoplasms). Capillarid nematodes may have intermediate or paratenic hosts. Using a laboratory transmission study, we confirmed that the parasite has a direct life cycle.

Rats fed either a cereal-based or purified diet of variable folate content (deficient, replete, or supplemented) inadvertently were infected with sialodacryoadenitis virus, which resulted in an increased frequency of hepatic mitochondrial DNA (mtDNA) deletions that persisted for three weeks after the period of acute signs of disease. The amount of the "common deletion" (4.8 kb, bases 8103–12937) in liver was measured by quantitative co-amplification of the mitochondrial D-loop and the mitochondrial deletion, using a real-time quantitative polymerase chain reaction assay. The relative abundance of mtDNA was determined by co-amplifying mitochondrial D-loop versus the rat β-actin gene. Virus-infected rats had more mtDNA deletions (P < 0.0001) and higher copy number (P < 0.0001) than did uninfected animals. There was no effect of diet on frequency of deletions. Diet affected mtDNA relative abundance in the infected, but not the uninfected rats. Relative abundance was higher (P = 0.004) in rats of the high folate group than in rats of the low-folate or folate-replete groups, and was significantly higher in rats of the cereal diet group than that in those of the purified diet group. In conclusion, sialodacryoadenitis virus infection in rats was associated with increased frequency of hepatic mtDNA deletions. Thus, sialodacryoadenitis virus infection mitigated biological processes in the liver of rats, and mtDNA damage was modulated by diet.

A large colony of laboratory zebrafish (Brachydanio rerio) used in the study of early vertebrate embryogenesis began experiencing acute, unexplained mortality that approached 100% among approximately 30-day-old resident fry. The initial differential diagnosis included ammonia, nitrite, or chlorine toxicosis, as well as iatrogenically induced toxicosis associated with improper sanitation procedures of laboratory equipment. Necropsy of dead and moribund fry prior to fixation revealed swarms of ovoid-shaped, motile, ciliated protozoa with a "spiraling football" motion. Wet mount preparations of various water samples also contained high numbers of similar protozoa. Histologic examination of affected fry revealed numerous, periodic acid-Schiff-positive forms within the body coelom, and epithelial and muscle tissues. The protozoa were consistent morphologically with members of the genus Tetrahymena, which is usually a free-living, nonpathogenic ciliated protozoa in fresh and saltwater environments. Relevant disease associated with Tetrahymena spp. in viviparous fish has been reported as a result of concurrent disease, immunosuppression, or poor water quality conditions. To the authors' knowledge, this is the first report of an epizootic involving laboratory maintained zebrafish, and the diagnostic course and therapeutic interventions undertaken to alleviate Tetrahymena species-associated clinical disease.

Severe type-3 von Willebrand's disease (vWD) was diagnosed in a young male rhesus monkey that had excessive bleeding from minor wounds. Plasma samples from the monkey had no detectable quantitative or functional von Willebrand factor (vWF), low Factor-VIII coagulant activity, and moderate prolongation of activated partial thromboplastin time. Testing of the affected monkey's extended family revealed a likely hereditary basis for the vWD, in that the sire and a paternal half-sister had markedly reduced plasma vWF concentration. Fresh whole blood was transfused to control frequent bleeding episodes throughout the monkey's life. Although vWD is the most common inherited bleeding disorder in humans and dogs, this is the first report of vWD in a nonhuman primate.