Specific Pathogen-free Colony Research Articles

Murine astrovirus (MuAstV) infection in mouse facilities in Brazil: First South American report.

This study aimed to investigate the presence of murine astrovirus (MuAstV) in Brazil. Fecal samples from mice belonging to four Brazilian animal facilities were collected and tested for MuAstV using real-time polymerase chain reaction. Of the 162 samples tested, 38 (23.5%) were positive for MuAstV, 33 (91.7%) of which came from specific-pathogen free colonies. Although most of the samples were obtained from asymptomatic animals, three mice presented diarrheal symptoms, and MuAstV was the only agent detected by molecular assay. Phylogenetic analysis revealed similarities between the MuAstV strains from this study and prototypes from the USA. MuAstV's high prevalence, environmental stability, genetic diversity and potential for persistent infections must be considered when evaluating health monitoring programs for laboratory rodents.

Microbiota and environmental health monitoring of mouse colonies by metagenomic shotgun sequencing

Metagenomic next-generation sequencing (mNGS) allows the monitoring of microbiota composition of murine colonies employed for scientific purposes in a single test by assessing the composition of gut microbiome and the detection of pathogens from fecal pellets. In this study, we tested the potential use of mNGS for monitoring both microbiota composition and the presence of pathogens through Environmental Health Monitoring, by using exhaust dust collection filters derived from individually ventilated cages (IVC) systems.mNGS analysis was performed on nucleic acids isolated from filters collecting air from the exhaust of: (1) cages with mice housed in a non-pathogen free facility; (2) animal-free cages with clean chow and bedding from the same facility; (3) cages housing mice from a specific-pathogen free (SPF) facility. mNGS results revealed correspondence between microbiome composition from fecal pellets and filter, including pathogenic bacteria (Helicobacter hepaticus, Helicobacter typhlonius, Chlamydia muridarum, Rodentibacter pneumotropicus, Citrobacter rodentium), intestinal protozoa (Tritrichomonas muris, Spironucleus muris) nematoda (Aspiculuris tetraptera) and eukaryotic parasites (Myocoptes musculinus), present in the colony. Entamoeba muris and Syphacia obvelata were detected in fecal pellets but not in filter. The animal free exhaust dust filter, exposed to clean cages (no mice) placed in the IVC after removal of all mice, exhibited the presence of the same pathogens due to contaminated connecting pipes, confirming the sensitivity of the approach. Conversely, the filter from SPF colony revealed the absence of pathogens.The current use of exhaust dust collection filters in health surveillance requires multiple molecular tests to identify specific pathogens and does not provide information on the colony microbiome. This work provides the proof-of-principle that assaying exhaust dust collection filters by mNGS for microbiota monitoring of laboratory mice is feasible. In its daily application, results suggest the usefulness of the test in SPF facilities, where pathogenic micro-organisms are expected to be absent. mNGS analysis of exhaust dust collection filters allows the analysis of multiple cages, reducing the number of tests required for pathogen detection and corresponding costs, and avoiding the use of sentinel mice.

Decontamination of genetically modified mice strains by embryo transfer for obtaining SPF colonies in a Brazilian animal facility

The introduction of new strains of mice in specific pathogen-free (SPF) animal facilities should be performed carefully to avoid breaking sanitary barriers. To meet this need, animals should be rederived to reduce infection risk and thus avoid research interference caused by loss of animal health status and welfare. The objective of this study was to implement mice embryo transfer in the laboratory mouse facility of the Department of Immunology at the Institute of Biomedical Sciences/University of São Paulo, Brazil. Embryo transfers were performed to rederive genetically modified mouse strains with undefined sanitary status, received from different research and educational institutions. Fertilized eggs at two-cell stage were obtained by natural means and transferred into the oviducts of SPF pseudo-pregnant female mice. All surgical procedures were performed under aseptic conditions. A total of 625 embryos were transferred into therecipients. 148 pups were born, of which 140 were reared. Viruses, bacteria and intestinal protozoa were eliminated using this technique. The improvement in the microbiological status of mice allowed their expansion in our SPF facility. With these results, we can stimulate the use of embryo transfer technique between rodent facilities in Brazil and thus encourage the distribution of better models to our scientific community.

Determination of major histocompatibility class I and class II genetic composition of the Caribbean Primate Center specific pathogen-free rhesus macaque (Macaca mulatta) colony based on massively parallel sequencing.

Knowledge of major histocompatibility complex (MHC) composition and distribution in rhesus macaque colonies is critical for management strategies that maximize the utility of this model for biomedical research. Variation within the Mamu-A and Mamu-B (class I) and DRB, DQA/B, and DPA/B (class II) regions of 379 animals from the Caribbean Primate Research Center's (CPRC) specific pathogen free (SPF) colony was examined using massively parallel sequencing. Analyses of the 7 MHC loci revealed a background of Indian origin with high levels of variation despite past genetic bottlenecks. All loci exhibited mutual linkage disequilibria while conforming to Hardy-Weinberg expectations suggesting the achievement of mutation-selection balance. The CPRC's SPF colony is a significant resource for research on AIDS and other infectious agents. Characterizing colony-wide MHC variability facilitates the breeding and selection of animals bearing desired haplotypes and increases the investigator's ability to understand the immune responses mounted by these animals.

The development of a specific pathogen free (SPF) barrier colony of marmosets (Callithrix jacchus) for aging research.

A specific pathogen free (SPF) barrier colony of breeding marmosets (Callithrix jacchus) was established at the Barshop Institute for Longevity and Aging Studies. Rodent and other animal models maintained as SPF barrier colonies have demonstrated improved health and lengthened lifespans enhancing the quality and repeatability of aging research. The marmosets were screened for two viruses and several bacterial pathogens prior to establishing the new SPF colony. Twelve founding animals successfully established a breeding colony with increased reproductive success, improved health parameters, and increased median lifespan when compared to a conventionally housed, open colony. The improved health and longevity of marmosets from the SPF barrier colony suggests that such management can be used to produce a unique resource for future studies of aging processes in a nonhuman primate model.

Immunologic detection of Giardia duodenalis in a specific pathogen-free captive olive baboon ( Papio cynocephalus anubis) colony.

Several commercial Giardia immunoassays were evaluated in baboons for sensitivity and specificity as well as ease of use in a large specific pathogen-free (SPF) colony. An additional objective was to identify the assemblage(s) of Giardia duodenalis present in this baboon colony. A direct immunofluorescent antibody test (IFAT) was used as the reference test. Tests evaluated were a patient-side rapid test for dogs and cats, a human rapid test, and a well-plate ELISA designed for use with humans. Test sensitivities and specificities were compared using the McNemar paired t-test and were further evaluated for agreement using an unweighted Cohen kappa statistic. When compared to the IFAT reference, both human tests were more sensitive than the veterinary test. Based on PCR and sequencing of the G. duodenalis small-subunit ribosomal RNA and glutamate dehydrogenase loci, assemblage AI was present in this baboon colony. We found that 10 of the 110 (9%) baboons in this SPF colony were infected with a zoonotic strain of G. duodenalis.

Specific pathogen free macaque colonies: a review of principles and recent advances for viral testing and colony management.

Specific pathogen free (SPF) macaques provide valuable animal models for biomedical research. In 1989, the National Center for Research Resources [now Office of Research Infrastructure Programs (ORIP)] of the National Institutes of Health initiated experimental research contracts to establish and maintain SPF colonies. The derivation and maintenance of SPF macaque colonies is a complex undertaking requiring knowledge of the biology of the agents for exclusion and normal physiology and behavior of macaques, application of the latest diagnostic technology, facilitiy management, and animal husbandry. This review provides information on the biology of the four viral agents targeted for exclusion in ORIP SPF macaque colonies, describes current state-of-the-art viral diagnostic algorithms, presents data from proficiency testing of diagnostic assays between laboratories at institutions participating in the ORIP SPF program, and outlines management strategies for maintaining the integrity of SPF colonies using results of diagnostic testing as a guide to decision making.

Establishment of a special pathogen free Chinese Wuzhishan Minipigs Colony.

To meet the increasing demands of specific pathogen free (SPF) minipigs in biomedical researches, 8 pregnant Chinese Wuzhishan minipigs (WZSP) sows with clear background were chosen to obtain SPF WZSP by hysterectomy. At 111 ± 2 days of the pregnancy, piglets were aseptically taken out from the sows and artificially suckled for 40 to 45 days in the positive isolators. Then, the piglets defined as F0 were transferred to barrier environment and fed with standard feeds. The original SPF colony was formed for breeding by selected piglets from F0 group of 6–8 months old. Biological characteristics of SPF WZSP were collected and further compared to those of conventional (CV) WZSP, including growth performance, reproductive performance, hematology and blood biochemistry, and major pathogens detection. As a result, 61 F0 piglets were obtained from 8 candidate sows, and 55 out of them survived. After strictly selection, 35 F0 piglets were used to form the original SPF colony, which produced 14 litters of SPF piglets defined as F1. Piglet survival rates, growth performance, and reproductive performance of SPF WZSP were similar to CV WZSP. Some hematology and blood biochemistry parameters showed significant differences between SPF and CV WZSP. Eighteen kinds of pathogens were identified to be free in F0 and F1 SPF colony by repeated pathogen detections. In conclusion, we established a satisfied SPF WZSP colony maintaining original characteristics, free of controlled diseases, and being proved to be a suitable laboratory animal.

Evaluation of the role of type I interferon to the development of type 1 diabetes. (BA3P.131)

Abstract Our goal is to identify the earliest immunologically relevant events that trigger autoimmune diabetes. The first detectable inflammatory signature in the NOD mouse is the upregulation of type I interferons and an interferon-inducible gene signature. We reason that understanding the early interferon signature may provide targets for the prevention, treatment, or diagnosis of autoimmune diabetes. The two ubiquitous interferon receptors (IFNAR and IFNGR) upregulate hundreds genes, some shared and some unique to each receptor. We backcrossed the type I interferon receptor onto NOD background to determine the relative contribution of IFNAR-inducible transcripts in type I diabetes induction. In our specific pathogen-free colony, NOD.IFNAR-/- mice developed diabetes at a decreased rate and penetrance from littermate controls. This reduction in incidence was gender independent and NOD.IFNAR heterozygous mice developed diabetes normally. There was a reduction in the number of the immunological infiltrates in the islets of Langerhans of NOD.IFNAR-/- mice from 4-8 wks of age as detected by flow cytometry and immunofluorescence. Concomitantly, there was a reduction in inflammatory gene expression in NOD.IFNAR-/- when compared to controls. Additionally, analysis of the NOD.IFNGR-/- mice showed a decrease in penetrance and kinetics of type I diabetes. Based on this data, we hypothesize that IFNAR and IFNGR induce a common genetic signature that is required for type I diabetes to proceed.

Are there advantages in the use of specific pathogen‐free baboons in pig organ xenotransplantation models?

Baboons have natural antibodies against pig antigens. We have investigated whether there are differences in anti-non-Gal pig antibody levels between baboons maintained under specific pathogen-free (SPF) conditions and those housed under conventional conditions (non-SPF) that might be associated with improved outcome after pig-to-baboon organ transplantation. Baboons (n=40) were housed indoors (SPF n=8) or in indoor/outdoor pens (non-SPF n=32) in colonies of similar size and structure. Non-SPF colonies harbor a number of pathogens common to non-human primate species, whereas many of these pathogens have been eliminated from the SPF colony. Complete blood cell counts (CBC), blood chemistry, and anti-non-Gal IgM and IgG levels were monitored. There were no significant differences in CBC or blood chemistry between SPF and non-SPF baboons. Anti-non-Gal IgM levels were significantly lower in the SPF baboons than in the non-SPF baboons (MFI 7.1 vs. 8.8, P<0.05). One SPF and two non-SPF baboons had an MFI >20; if these three baboons are omitted, the mean MFIs were 4.8 (SPF) vs. 7.5 (non-SPF) (P<0.05). Anti-non-Gal IgG was minimal in both groups (MFI 1.0 vs. 1.0). As their levels of anti-non-Gal IgM are lower, baboons maintained under SPF conditions may be beneficial for xenotransplantation studies as the initial binding of anti-pig IgM to an α1,3-galactosyltransferase gene-knockout pig organ may be less, thus resulting in less complement and/or endothelial cell activation. However, even under identical SPF conditions, an occasional baboon will express a high level of anti-non-Gal IgM, the reason for which remains uncertain.

Isolation, characterization and microincapsulation of neonatal porcine Sertoli cells obtained from a specific pathogen free (SPF) herd

Porcine Sertoli cells (pSC) have been successfully employed as cell therapy in pre-clinical studies of several immune-based and chronic degenerative diseases. In order to prevent any transmission of infectious adventitious agents to the cells graft recipients, we have set up, according to our previously described method (Luca et al., 2007) pSC monolayers obtained from specific pathogen free (SPF) certified neonatal pigs, born in the unique SPF colony in Italy. pSC were assessed and characterized as far as viability, by ethidium bromide and fluorescein diacetate (EB/FD), Mullerian inhibiting substance (AMH), and insulin-like 3 (INSL3), alpha-smooth muscle actin (ASMI) both by immunofluorescence (IF) and cytofluorimetric analysis (CA) were concerned. pSC were encapsulated in alginate microcapsules (MCpSC), with MCp- SC functional competence and biocompatibility being determined both in vitro, by AMH, inhibin B, TGF-beta, IGF-I secretion and in vivo in experimental animal models, respectively. Results demonstrated the high purity of our pSC monolayers (95% of AMH+cells), with negligible contamination by Leydig (2%) and peritubular cells (3%). Microencapsulation did not alter pSC viability and even after 4 months postimplantation, all the retrieved microcapsules retained morphology and function. In conclusion, we have uniquely obtained, from a SPF herd, highly purified, viable and functional pSC that might poten-tially apply to humans.

Abstract A100: Helicobacter hepaticus contributes to mammary gland carcinogenesis through bacterial translocation and subsequent expansion of cancer-promoting myeloid-derived suppressor cells

Abstract Introduction: Gastrointestinal (GI) tract dysbiosis and resulting inflammation are implicated in breast carcinogenesis. CD11b+GR1+ myeloid- derived suppressor cells (MDSCs) are important drivers of breast cancer. Here we report that in APCMin/+ mice treated with enteric Helicobacter hepaticus (H. hepaticus) MDSCs cells are dramatically expanded and, along with the pathogen H. hepaticus, are directly recruited to mammary gland lymph node, where they suppress anti-carcinogenic cytotoxic T-cells and promote procarcinogenic inflammation, respectively. Methods: In our H. hepaticus and specific pathogen free colony we inoculated female APCMin/+ mice with H. hepaticus and compared neoplastic and inflammatory outcomes to the non-infected controls. Results: Successful infection was confirmed by H. hepaticus specific qPCR and fluorescence in situ hybridization (FISH). H. hepaticus was identified not only within the small intestine but also within the mammary gland lymph node and mammary tumor tissue of the treated group. In APCMin/+ mice treated with chow diet, H. hepaticus-induced procarcinogenic CD11b+GR1+ cells within the lamina propria of the small intestine, the surrounding mesenteric lymph nodes, the spleen, peripheral blood and the mammary gland lymph nodes. In H. hepaticus infected mice, this was accompanied by increased splenic expression of IL-1β, IL-6 and GM-CSF and the increased expression of IL-1β, IL-6 and GM-CSF, IL-23a and IL-17a in the small intestine. Interestingly, adding high fat diet to H. hepaticus infected mice appeared to further promote the trafficking of MDSCs from the spleen into the peripheral blood and regional lymph nodes with increased serum inflammatory cytokines production cytokines, including IL-6. This appears to have been mediated, at least in part, by adipocyte signaling to myeloid precursors. Conclusions: We show the presence of H. hepaticus in mammary gland and intestine, which correlated with mammary gland tumor incidence and increased intestinal tumor multiplicity. The co-existence of enteric H. hepaticus infection appears to promote mammary tumorigenesis through upregulation of IL-1β, IL-6 and GM-CSF and the resulting trafficking of tumor promoting CD11b+GR1+ MDSCs. We are currently examining the route and broader relevance of H. hepaticus extra-intestinal translocation and whether this has direct effects on tumorigenesis, consistent with findings in other Helicobacter spp. Citation Format: Xiaowei Chen, Daniel L. Worthley, Zhongming Ge, Yagnesh Tailor, Christian Kaufman, Lenzie Cheaney, Lesley Kline, Gladys Y. Valeriano, Christoph B. Westphalen, Susan E. Erdman, James G. Fox, Timothy C. Wang. Helicobacter hepaticus contributes to mammary gland carcinogenesis through bacterial translocation and subsequent expansion of cancer-promoting myeloid-derived suppressor cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A100.

Evaluation of reproduction and raising offspring in a nursery-reared SPF baboon (Papio hamadryas anubis) colony.

Baboons (Papio hamadryas anubis) of a conventional breeding colony were nursery-reared to create a specific pathogen-free (SPF) baboon-breeding program. Because the founding generations were nursery-reared until 2 years of age, it was suspected that the SPF baboons would exhibit increased reproductive challenges as adults. Mothering behavior was of interest, because SPF females were not exposed to parental role models during the nursery-rearing process. We compared reproductive data from the SPF baboon breeding program during its first 10 years with data from age-matched baboons during the same period from an established, genetically-similar conventional breeding colony. We also evaluated records documenting mother-infant behaviors within the SPF colony. The average age of menarche in SPF females was 3.3 years. The overall live birth rate of both SPF and conventional females was approximately 90%, with no difference in pregnancy outcome between the two colonies. The average age at first conception for SPF females was earlier (4.2 years) than that of the conventional females (4.7 years). In both colonies, primiparous females were more likely to abort than multiparous females. Similarly, primiparous females were more likely to lose their infants to death or human intervention. A mothering score system was developed in the SPF colony to facilitate intervention of poor mother-infant relationships. Records revealed 70% of SPF mothers were able to raise one or more of their infants successfully to at least 180 days of age, which did not differ from conventional mothers. SPF females returned to post-partum amenorrhea 27 days sooner on average than the conventional females, independent of dam age. The nursery-rearing process used for recruitment into the SPF colony therefore did not have an adverse effect on reproduction or rearing offspring.

Specific‐pathogen‐free status is associated with lower infant mortality rate in rhesus macaque (Macaca mulatta) colonies at the California National Primate Research Center

Specific-pathogen-free (SPF) rhesus macaques, Macaca mulatta, are a valuable resource in biomedical research, and demographic analysis plays a significant role in colony management. Data collection included SPF levels, gender, birth year, season of birth, birth location, rearing condition, maternal pregnancy history, and maternal age. Infant mortality in SPF rhesus macaques was compared with that in non-SPF rhesus macaques at the California National Primate Research Center over a six-year period, using Cox proportional regression analysis. In infants born to multiparous dams, the SPF infants had a significantly lower rate of mortality than non-SPF infants. There was no statistically significant difference in infant mortality between different SPF levels. Elimination of selected endemic viruses from breeding populations of rhesus macaques for the purpose of SPF colony development is associated with a significant reduction in the infant mortality rate.

Genetic characterization of specific pathogen-free rhesus macaque (Macaca mulatta) populations at the California National Primate Research Center (CNPRC).

A study based on 14 STRs was conducted to understand intergenerational genetic changes that have occurred within the California National Primate Research Center's (CNPRC) regular specific pathogen-free (SPF) and super-SPF captive rhesus macaque populations relative to their conventional founders. Intergenerational genetic drift has caused age cohorts of each study population, especially within the conventional population, to become increasingly differentiated from each other and from their founders. Although there is still only minimal stratification between the conventional population and either of the two SPF populations, separate derivation of the regular and super-SPF animals from their conventional founders has caused the two SPF populations to remain marginally different from each other. The regular SPF and, especially, the super-SPF populations have been influenced by the effects of differential ancestry, sampling, and lost rare alleles, causing a substantial degree of genetic divergence between these subpopulations. The country of origin of founders is the principal determinant of the MHC haplotype composition of the SPF stocks at the CNPRC. Selection of SPF colony breeders bearing desired genotypes of Mamu-A*01 or -B*01 has not affected the overall genetic heterogeneity of the conventional and the SPF research stocks.Because misclassifying the ancestry of research stocks can undermine experimental outcomes by excluding animals with regional-specific genotypes or phenotypes of importance, understanding founder/descendent genetic relationships is crucial for investigating candidate genes with distinct geographic origins. Together with demographic management, population genetic assessments of SPF colonies can curtail excessive phenotypic variation among the study stocks and facilitate successful production goals.

Specific Pathogen-Free Macaques: Definition, History, and Current Production

Specific pathogen-free (SPF) macaque colonies are now requested frequently as a resource for research. Such colonies were originally conceived as a means to cull diseased animals from research-dedicated colonies, with the goal of eliminating debilitating or fatal infectious agents from the colony to improve the reproductive capacity of captive research animals. The initial pathogen of concern was Mycobacterium tuberculosis (M.tb.), recognized for many years as a pathogen of nonhuman primates as well as a human health target. More recently attention has focused on four viral pathogens as the basis for an SPF colony: simian type D retrovirus (SRV), simian immunodeficiency virus (SIV), simian T cell lymphotropic/leukemia virus (STLV), and Cercopithecine herpesvirus 1 (CHV-1). New technologies, breeding, and maintenance schemes have emerged to develop and provide SPF primates for research. In this review we focus on the nonhuman primates (NHPs) most common to North American NHP research facilities, Asian macaques, and the most common current research application of these animals, modeling of human AIDS.

α7 Nicotinic Receptor Gene Promoter Polymorphisms in Inbred Mice Affect Expression in a Cell Type-specific Fashion

Inbred mouse strains display significant differences in their levels of brain alpha7 nicotinic acetylcholine receptor (alpha7 nAChR) expression, as measured by binding of the alpha7-selective antagonist alpha-bungarotoxin. Variations in alpha-bungarotoxin binding have been shown to correlate with an animal's sensitivity to nicotine-induced seizures and sensory gating. In two inbred mouse strains, C3H/2Ibg (C3H) and DBA/2Ibg (DBA/2), the inter-strain binding differences are linked to a restriction length polymorphism in the alpha7 nAChR gene, Chrna7. Despite this finding, the molecular mechanism(s) through which genetic variability in Chrna7 may contribute to alpha7 nAChR expression differences remains unknown. However, studies of the human alpha7 nAChR gene (CHRNA7) previously have demonstrated that CHRNA7 promoter polymorphisms are associated with differences in promoter activity as well as differences in sensory processing. In the present study, a 947-base pair region of the Chrna7 promoter was cloned from both the C3H and DBA/2 inbred mouse strains in an attempt to identify polymorphisms that may underlie alpha7 nAChR differential expression. Sequence analysis of these fragments identified 14 single nucleotide polymorphisms (SNPs). A combination of two of these SNPs affects promoter activity in an in vitro luciferase reporter assay. These results suggest a mechanism through which the Chrna7 promoter genotype may influence interstrain variations in alpha7 nAChR expression.

Retroviremia in Commercial Pigs and Its Preliminary Association with Poor Health

Retroviremia in Commercial Pigs and Its Preliminary Association with Poor Health

Blood copper concentrations and cuproenzyme activities in a colony of cats.

There is little published information on plasma copper and whole blood copper concentrations in cats, and except for extracellular superoxide dismutase (EC SOD), we are unaware of published values for cat cuproenzyme activities. In this study we determined plasma and whole blood copper concentrations, plasma ceruloplasmin (CP), EC SOD and diamine oxidase (DAO) activities, and erythrocyte superoxide dismutase (RBC SOD) activity in a specific pathogen-free colony of cats. Results were evaluated for differences based on age and sex. Blood was obtained from 20 adult cats (10 males, 10 females; >1 year of age) and 20 immature cats (10 males, 10 females; <1 year of age). Copper concentrations were determined using atomic absorption spectrophotometry. EC SOD and RBC SOD activities were determined using the pyrogallol oxidation method, and CP activity was determined by the oxidation of 0 dianisidine dihydrochloride. DAO activity was measured using a colorimetric assay. Differences among groups were analyzed using ANOVA. There were significant differences in mean plasma copper concentration, and CP and DAO activities (P=.001,.0001, and.02, respectively) among the 4 groups of cats. No age differences were identified. Male cats had significantly greater mean (+/- SEM) plasma copper (15.4 +/- 0.9 micromol/L versus 11.3 +/- 0.6 micromol/L; P =.001) concentrations, and CP (66.8 +/- 2.9 U/L versus 39.7 +/- 2.1 U/L; P=.0001) and DAO (6.68 +/- 0.16 U/L versus 6.15 +/- 0.1 U/L; P =.03) activities compared with female cats. Males had significantly greater whole blood copper concentrations (16.16 +/- 0.55 micromol/L versus 13.36 +/- 0.52 micromol/L; P =.002) than female cats. Differences exist between male and female cats with respect to blood copper concentrations, and CP and DAO activities.

Establishment of specific pathogen-free guinea-pig colonies using limited-flora guinea-pigs associated with conventional guinea-pig flora, and monitoring of their cecal flora.

Six groups of limited flora (LF) Hartley guinea-pigs were produced by inoculation of hysterectomy-derived GF guinea-pigs with various combinations of cecal bacteria of conventional (CV) guinea-pigs to determine the effective bacterial cocktails for the establishment of a specific pathogen free (SPF) colony. Bifidobacterium magnum (Bif) isolated from CV guinea-pigs was used for pretreatment. The mortality of LF guinea-pigs inoculated with only Bif was 75%, and that of those inoculated with Bif plus chloroform-treated cecal suspension (CHF) or Bif plus CHF plus 32 isolates from CV guinea-pigs was 40 to 66.7%. These three groups were in an unhealthy condition with mucoid enteritis-like diarrhea. However, the mortality of LF guinea-pigs inoculated with the anaerobic growth on EG plates injected with 10(-5) dilution of cecal contents (CF) or inoculated with Bif plus CF was 6.3 and 15%, respectively. These latter two groups of LF guinea-pigs were transferred to separate barrier rooms and some of the LF guinea-pigs were maintained in isolators as a source of intestinal flora for SPF guinea-pigs. The composition of cecal flora of LF guinea-pigs was stable for a long time, and bacteroidaceae and peptococcaceae were maintained as predominant components. The basic composition of the cecal flora of SPF guinea-pigs originated from LF guinea-pigs, which consists mainly of the anaerobic bacteria, was not changed over a long period, and the flora composition became similar to that in CV guinea-pigs. Guinea-pig-specific pathogens from the SPF colonies were not detected during experiments.