The use of laboratory mice to investigate correlates of infectious disease, including infection kinetics, cellular alterations, cytokine profiles, and immune response in the context of an intact host has expanded exponentially in the last decade. A marked increase in the availability of transgenic mice and research tools developed specifically for the mouse parallels and enhances this research. Human granulocytic ehrlichiosis (HGE) is an emerging, zoonotic disease caused by tick-borne bacteria. The HGE agent (Anaplasma phagocytophila) is one of two recognized pathogens to cause human granulocytic ehrlichiosis (HGE). The mouse model of HGE complements in vitro tissue culture studies, limited in vivo large animal studies, and ex vivo studies of human and ruminant neutrophils, and promises new avenues to approach mechanisms of disease. In the overview reported here, we focus principally on current research into HGE pathogenesis using the mouse model. Included is a discussion of current changes in ehrlichial classification and nomenclature, a review of ehrlichial biology and ecology, and highlights of clinical disease in animals and people.

Genetic management is required to maintain genetic diversity by minimizing inbreeding and genetic subdivision in colonies of animals bred for biomedical research. Polymorphic short tandem repeat (STR) loci are useful for genetic management because they facilitate parentage assignments, genetic characterization of individuals, and estimates of baseline population genetic parameters. Using highly informative STR loci, we estimated gene diversity and F-statistics to determine the level of genetic heterogeneity and genetic structure of three specific-pathogen-free (SPF) rhesus macaque (Macaca mulatta) colonies. Effective population sizes, variance in male reproductive success, and rate of decrease in genetic variability also were estimated for two of the three colonies.We documented the overall success of genetic management in maintaining genetic diversity in captive colonies. We report that even genetically managed SPF colonies, despite maintaining high and stable levels of gene diversity (over 0.75), are prone to genetic subdivision due to different management strategies, founder effects, genetic isolation, and drift. These processes are accelerated by the high variances in male reproductive success and low adult sex ratios that are typical of captive rhesus macaque breeding groups, both of which reduce the effective population sizes of these groups.

Purpose: The purpose of the study reported here was to determine the relationship of bispectral index (BIS) to multiples of sevoflurane minimal alveolar concentration (MAC), using proprietary patch or subdermal needle electrodes in dogs.Methods: Eight English Pointers (4 males, 4 females; mean ± SD age and body weight of 3.9 ± 2.2 years and 20.7 ± 4.1 kg, respectively) were studied. Sevoflurane MAC was determined in each dog, using the standard tail clamp technique. One week later, BIS was determined in each dog at 0.8, 1.0, 1.5, and 2.0 MAC multiples of sevoflurane, using proprietary patch electrodes and 29-gauge platinum needle electrodes applied in randomized order. Ventilation was controlled, and atracurium (0.2 mg/kg of body weight, followed by 6 μg/kg/min, i.v.) was administered to eliminate the electromyographic artifact from the electroencephalogram. The BIS was determined, using an A-2000 BIS monitor connected to a computer for data logging at five-second intervals. After a 15-min equilibration period at each sevoflurane MAC-multiple, BIS data were collected for five minutes, and median BIS values were calculated. Heart rate, direct mean arterial blood pressure, esophageal temperature, and arterial pH and blood gas tensions were measured immediately after each BIS collection period. End-tidal CO₂ and sevoflurane concentrations were continuously monitored, using an infrared gas analyzer. Data were analyzed, using one-way repeated measures analysis of variance (P < 0.05). Agreement of BIS values from each electrode type was determined.Results: Mean ± SD sevoflurane MAC was 2.1 ± 0.3%. Mean ± SD BIS values at 0.8, 1.0, 1.5, and 2.0 MAC were 77 ± 3, 73 ± 5, 57 ± 7, and 53 ± 7, respectively, for patch electrodes and 80 ± 6, 72 ± 7, 56 ± 4, and 50 ± 5, respectively, for subdermal needle electrodes. At 2 MAC, BIS could not be determined in six dogs due to presence of burst suppression in the EEG. The regression equation comparing electrodes was: BIS (subdermal) = −5.5 + (1.1 × BIS [patch]); R² = 0.846; bias = −0.192, with a 95% confidence interval of −9.96 to 9.56.Of the other measured variables, none were significantly different between electrode types. Within each group of electrode type, MAP was significantly different among MAC multiples. Within the patch electrode group, PaO₂, bicarbonate concentration, and base excess were significantly different among MAC multiples.Conclusions: Bispectral index significantly decreased with increasing sevoflurane MAC multiples over the range of 0.8 to 2.0 MAC, using patch or subdermal electrodes in dogs. Use of subdermal needle electrodes is a reliable and practical alternative to use of patch electrodes for measurement of BIS in dogs.

The mucosal immune system interacts with the external environment. In the study reported here, we found that bedding materials can influence the intestinal immune responses of mice. We observed that mice housed on wood, compared with cotton bedding, had increased numbers of Peyer's patches (PP) visible under a dissecting microscope. In addition, culture of lymphoid organs revealed increased production of total and virus-specific IgA by PP and mesenteric lymph node (MLN) lymphocytes from mice housed on wood, compared with cotton bedding. However, bedding type did not influence serum virus-specific antibody responses. These observations indicate that bedding type influences the intestinal immune system and suggest that this issue should be considered by mucosal immunologists and personnel at animal care facilities.

Purpose: The pathogenesis of polycystic kidney disease (PKD) has not been firmly established; however, our current knowledge of cystogenesis and human cystic disease has been greatly advanced by a variety of animal models of PKD. To study the onset and degree of cyst formation in PKD mouse models without requiring sacrifice of these animals, we have initiated magnetic resonance imaging (MRI) studies of the juvenile cystic kidney (jck) mouse model.Methods: The MRI experiments were performed by use of a Bruker 8.5 T system, on seven-week-old mice that were homozygous for the recessive jck mutation and that manifested PKD. Kidney volume was measured, using three-dimensional segmentation postprocessing techniques.Results: The MR images of the enlarged kidneys from affected mice had regions of high signal intensity, with a radial distribution, which reflected the dilated collecting ducts observed in the corresponding histologic slices. The volume of PKD-affected kidney was about 4 times greater than that of the normal kidney.Conclusions: Magnetic resonance imaging has the ability to non-invasively assess cystic disease in mouse models of PKD. Of considerable importance is the opportunity to characterize this disease without sacrificing the animal. The three-dimensional MRI segmentation method provides accurate calculation of renal volume.

Purpose: To develop a polymerase chain reaction (PCR) assay for specific detection of Citrobacter rodentium in fecal samples of mice and to compare this assay with bacterial isolation and identification methods.Methods: The target sequence of the PCR assay was the espB gene encoding a secreted virulence factor. To facilitate visual identification during primary isolation on MacConkey agar containing ampicillin, C. rodentium ATCC type strain 51459 was transformed by use of a plasmid encoding the enhanced green fluorescent protein (EGFP) and ampicillin resistance. The EGFP-C. rodentium was inoculated into Swiss Webster (SW) mice to study the time course of detection of the organism by use of fecal PCR analysis, bacterial isolation, and development of colonic hyperplasia by light microscopy. Lactose-fermenting fluorescent bacterial colonies identified during primary isolation of fecal bacteria on MacConkey-ampicillin agar were identified by use of biochemical typing.Results: Mice inoculated with EGFP-transformed C. rodentium developed colonic mucosal hyperplasia, characterized by a three-fold increase in colonic crypt height that peaked at post-inoculation day (PID) 14. The espB PCR assay detected as little as 0.3 colony-forming units of C. rodentium. The PCR assay was specific in that it did not detect the espB gene of Escherichia coli O157. Results of in vivo studies in SW mice indicated that EGFP-C. rodentium could be detected by use of espB fecal PCR analysis in 100% of inoculated mice tested on PID 1, 3, 7, and 8, in 60% on PID 9, and in 20% on PID 10 (n = 5). Bacterial isolation from the same fecal samples detected the organism in 100% of the inoculated mice on PID 7, in 50% on PID 8, and in none on subsequent PID 9–14. The ability of the PCR assay to detect C. rodentium in fresh feces of inoculated mice was significantly better than that of bacterial isolation methods (Fisher-Irwin exact test, P < 0.01). At the time of peak colonic hyperplasia, the organism could no longer be cultivated or detected in mice by use of fecal PCR analysis.Conclusions: The EGFP-C. rodentium was capable of inducing transmissible murine colonic hyperplasia similar to that previously reported in SW mice. The PCR assay for detection of the espB gene sequence of C. rodentium in total fecal DNA was a more sensitive diagnostic assay than was bacterial isolation.

We have established safe and efficient methods for autologous hematopoietic stem cell (HSC) transplantation in cynomolgus monkeys (Macaca fascicularis) that include regimens of supportive care to ensure survival during hematopoietic reconstitution following otherwise lethal total body irradiation. Eleven young adult cynomolgus monkeys were studied. Bone marrow was aspirated from the ilium and/or tuber ischiae after administration of recombinant human stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF). Using the immunomagnetic selection method, CD34⁺ cells were then isolated (90 to 95% pure) as a fraction containing HSCs. Just prior to transplantation, the animals received myeloablative total body irradiation—500 to 550 cGy daily for two days. The monkeys re-infused with CD34⁺ cells developed moderate to severe myelosuppression, with some animals requiring intravenous hyperalimentation, antibiotic administration, and blood transfusion. Hematopoiesis was restored in all animals after transplantation. It took 12 days, on average, until the peripheral white blood cell count reached more than 1,000 cells/μl. Up to two years after transplantation, signs of radiation-induced pneumonitis or other radiation-related disorders were not evident at the aforementioned dose of irradiation. This transplantation model will be useful for testing new approaches using HSCs for therapy of many diseases and will offer unique insights into the biology of these cells.

Maternal administration of perphenazine decreased the incidence of cannibalism in colonies of interferon-γ, interleukin (IL-4, IL-10, and IL-12) knockout mice of the DBA/1 and C57BL/6 background strains. This colony had high incidence of neonatal death due to cannibalism in approximately 50% of the pups. Perphenazine was administered to the dams in the drinking water, beginning on the day before or the morning of parturition. The medicated water was supplied at two dosages: 0.5 mg/ml and 0.025 mg/ml, resulting in a dosage of 4 mg/kg of body weight and 2 mg/kg, respectively, to the dams. Dams that were treated with perphenazine weaned 76.4% of their pups, compared with non-treated dams that weaned only 59.4% of their pups. Timing of the administration of perphenazine did not have a significant impact on efficacy; also, both doses were equally effective at preventing cannibalism. These findings indicate that perphenazine can modify poor maternal behavior such as cannibalism, resulting in more efficient production of valuable knockout mice.

Elimination of an enzootic infection of mouse hepatitis virus (MHV) from a large population of genetically engineered mice was accomplished by selecting seropositive, non-infective breeders for a newly restored MHV-free breeding colony. An ELISA was used to test for the presence of MHV-specific antibody, and TaqMan reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was used to detect MHV in the feces. After 10 weeks of intentional exposure, approximately 30% of mice with MHV antibodies continued to shed MHV in the feces. A natural transmission study was conducted to validate that positive fecal RT-PCR results indicated presence of infective virus. Sentinel results from the re-instituted breeding colony indicated that MHV was successfully eliminated by use of RT-PCR analysis for selection of non-infective mice.

The finding of Helicobacter hepaticus infection in our acetylcholinesterase (AChE) knockout mouse colony led to a search for a treatment. One-hundred percent of AChE +/+, 100% of AChE +/−, and 35% of AChE −/− mice tested positive. The lower infection rate in AChE −/− mice, who are routinely weaned on day 15, suggested that early weaning might be an effective eradication strategy. The AChE +/+ and +/− mice were weaned on days 13, 14, 15, or 16. Litters were placed in sterile, heated, isolator cages. Animals were fed liquid Ensure Fiber and 11% fat pelleted diet. Feces were tested for the presence of H. hepaticus by use of DNA amplification. Litters weaned on days 14, 15, or 16 had a high rate (68, 63, and 100%, respectively), whereas litters weaned on day 13 had a lower (8%) rate of infection. Uninfected animals have remained free of H. hepaticus through day 120. Pups weaned on day 13 lost body weight, beginning on day 14, but recovered by day 16. It is concluded that the non-coprophagic behavior of AChE −/− mice accounts for a low infection rate and that the combination of early weaning, routine testing, and culling provide an effective method for eradication of H. hepaticus.

Measles virus (MV), a highly infective paramyxovirus, has caused sporadic epizootics characterized by high morbidity and increased mortality in nonhuman primates. Measles vaccines for human use, although effective, are cost prohibitive for use in primate colonies. We compared the efficacy of one or two doses of Vanguard D-M, a canine distemper-measles (CD-M) vaccine, with a single dose of Attenuvax, a human measles vaccine. Compared with 81% of animals inoculated with Attenuvax, all animals inoculated with one or two doses of Vanguard developed detectable MV antibodies. One year after immunization, six juveniles from each vaccine group, along with three unvaccinated controls, were challenged with pathogenic MV and were monitored for clinical signs of disease, viremia, viral shedding, and immune response. All uninoculated controls developed clinical disease and viremia, and shed virus in nasopharangeal secretions. Subclinical viremia without viral shedding was identified in two Attenuvax- and two single-dose Vanguard-inoculated animals. Viremia was not detected in any two-dose Vanguard-inoculated animals. Significantly higher neutralization antibody titers were observed in animals receiving Vanguard. Results of this study indicate that Vanguard is at least as efficacious as Attenuvax for protection of rhesus macaques. The considerably lower cost of Vanguard makes vaccination against measles in large breeding colonies economically feasible.