An attempt to eradicate Herpesvirus simiae from a rhesus monkey breeding colony.

In the fall of 1987 an attempt to establish a Herpesvirus simiae (B-virus)-negative rhesus monkey (Macaca mulatta) breeding colony was initiated at the Armstrong Laboratory. A serologic testing program was used to identify all monkeys into groups that were either positive or negative to B-virus based on serologic tests. Segregation of the groups allowed the creation of breeding harems that were exclusively seropositive or -negative to B-virus. Animals that were serologically positive were kept in breeding to maintain infant production levels not unlike those previous to segregation. Decreasing numbers of animals converted to a positive status during the first three serum tests for B-virus in the program. During 1990, an increase in the number of monkeys converting to positive status and the discovery of an indeterminate status demonstrated that latency of B-virus in the rhesus may have the potential to defeat an eradication attempt not conscientiously pursued.     

B-virus specific-pathogen-free breeding colonies of macaques (Macaca mulatta): retrospective study of seven years of testing.

BACKGROUND AND PURPOSE: National Institutes of Health's Division of Comparative Medicine has sponsored a multi-institutional program for the establishment of specific-pathogen-free (SPF) macaque colonies. B virus (Herpesvirus simiae, Cercopithecine herpesvirus type 1) has been targeted in this surveillance. Participating institutions have established individual timetables for frequency of testing and types of monitoring and husbandry techniques, all with the common goal of producing pathogen-free monkeys for research. The greatest biosecurity threat to the program comes from failure to detect seronegative latent infections, either in first-year macaques or macaques introduced in subsequent years, although these are supposed to operate as closed colonies. METHODS: From January 1990 through December 1996, we screened macaques for B virus, using enzyme-linked immunoabsorbant assay (ELISA) and Western blot analysis. RESULTS: During the first year, 1,097 macaques from six colonies were tested, and 88.4% tested negative for B virus. During the seventh year, 1,843 were tested, of which 99.7% tested negative. Seropositive macaques were detected as late as the seventh year. CONCLUSIONS: An aggressive program to establish an SPF colony of captive breeding macaques can be effective in reducing the risk of B-virus exposure.     

Drosophila SPF45: a bifunctional protein with roles in both splicing and DNA repair

The sequence of the SPF45 protein is significantly conserved, yet functional studies have identified it as a splicing factor in animal cells and as a DNA-repair protein in plants. Using a combined genetic and biochemical approach to investigate this apparent functional discrepancy, we unify and validate both of these studies by demonstrating that the Drosophila melanogaster protein is bifunctional, with independent functions in DNA repair and splicing. We find that SPF45 associates with the U2 snRNP and that mutations that remove the C-terminal end of the protein disrupt this interaction. Although animals carrying this mutation are viable, they are nevertheless compromised in their ability to regulate Sex-lethal splicing, demonstrating that Sex-lethal is an important physiological target of SPF45. Furthermore, these mutant animals exhibit phenotypes diagnostic of difficulties in recovering from exogenously induced DNA damage. The conclusion that SPF45 functions in the DNA-repair pathway is strengthened by finding both genetic and physical interactions between SPF45 and RAD201, a previously uncharacterized member of the RecA/Rad51 protein family. Together with our finding that the fly SPF45 protein increases the survival rate of mutagen-treated bacteria lacking the RecG helicase, these studies provide the tantalizing suggestion that SPF45 has an ancient and evolutionarily conserved role in DNA repair.     

湖南省2006～2010年实验动物微生物质量监测结果分析

目的了解湖南省实验动物微生物学质量,为实验动物实行科学管理提供重要依据,保证实验动物质量,确保实验结果准确和实验动物科技事业工作者的职业健康与安全。方法在相关单位生产繁殖群以单纯随机抽样原则采样,按照GB 14922.2-2001、GB/T 14926-2001和GB/T14926.21-2008进行实验动物微生物检测。结果 SPF级大鼠批次合格率为73.68%,SPF级小鼠批次合格率为77.78%,普通级新西兰兔批次合格率为60.61%,普通级豚鼠批次合格率为100%。结论我省2006～2010年实验动物微生物学质量前期呈现下降趋势,后期得到较大改善。要继续采取措施加强实验动物管理,消除实验动物种群微生物感染。     

Development of Breeding Populations of Rhesus Macaques (Macaca mulatta) That Are Specific Pathogen-free for Rhesus Cytomegalovirus

Development of breeding colonies of rhesus macaques (Macaca mulatta) that are specific pathogen-free (SPF) for rhesus cytomegalovirus (RhCMV) is relatively straightforward and requires few modifications from current SPF programs. Infants separated from the dam at or within a few days of birth and cohoused with similarly treated animals remain RhCMV seronegative indefinitely, provided they are never directly or indirectly exposed to a RhCMV-infected monkey. By systematically cohousing seronegative animals into larger social cohorts, breeding populations of animals SPF for RhCMV can be established. The additional costs involved in expanding the current definition of SPF status to include RhCMV are incremental compared with the money already being spent on existing SPF efforts. Moreover, the large increase in research opportunities available for RhCMV-free animals arguably would far exceed the development costs. Potential new areas of research and further expansion of existing research efforts involving these newly defined SPF animals would have direct implications for improvements in human health.Abbreviations: HCMV, human cytomegalovirus; NHP, nonhuman primate; RhCMV, rhesus cytomegalovirus; SPF, specific pathogen-freeThe impetus for expanding the current SPF definition to include RhCMV is 2-fold. The first is the increasing number of studies involving infection of rhesus macaques with RhCMV as a nonhuman primate (NHP) model of human infection with human cytomegalovirus (HCMV). The second is the recognition that the current SPF protocols result in animals that are also uninfected with RhCMV, such that a relatively minor change in derivation can generate monkeys that meet the current SPF definition and that are uninfected with other endemic viruses, including RhCMV and simian foamy virus.     

Intestinal microflora functions in laboratory mice claimed to harbor a “normal” intestinal microflora. Is the SPF concept running out of date?

For many years, laboratory animal breeders have used a mixture of eight bacterial strains, the so-called Altered Schaedler Flora (ASF) to inoculate Caesarian derived offspring when establishing colonies of Specific Pathogen Free (SPF) rodents fulfilling the criteria worked out by regulatory agencies as AALAS, FELASA, etc. However, recently it was shown in this journal that such SPF animals harbored a fecal flora far different from that of feral mice.Over the years, we have worked with functional aspects of host-microbe interactions(s) and the aim of the present study was to analyze some intestinal microbial biochemical activities in mice harboring an ASF flora. In the five parameter studied, the ASF mice showed a pattern similar to what is found in germfree mice and rats, demonstrating an absence of microorganisms capable of performing these reactions. These findings call for a re-considering of the SPF concept. Presence of important microbiological functions should be taken into consideration when rodents are used in biomedical research.     

Comparison of fecal biota from specific pathogen free and feral mice

Specific pathogen free (SPF) rodents are derived from germfree animals that are colonized with Schaedler's flora, a cocktail of eight bacterial strains isolated from the natural biota of mice. During successive generations SPF animals acquire a complex biota, but it is not known how similar it is to natural mouse biota. Therefore, fecal pellets of two feral mice and three SPF mice were studied by small subunit ribosomal DNA sequence analysis. After amplification of 16S rDNA by Bacterial Kingdom-specific primers, 132 rDNA clones from feral mice and 219 clones from SPF mice were placed phylogenetically. Forty-four percent of recovered rDNAs from feral mice were from organisms belonging to the Ribosomal Database Project's Bacteroides Group with significant proportions also coming from lactobacilli, the Clostridium coccoides Group and the Clostridium leptum Group. Although the SPF biota appeared equally complex at lower phylogenetic levels, the major phylogenetic groups represented were less diverse in that 92% of rDNA's from SPF mice mapped to groups of clostridia with 79% to the C. coccoides Group alone. Given the number of physiological parameters influenced by the gut biota and the importance of mice in biomedical research, further investigations are warranted.     

Immunological function of food-restricted germfree and specific pathogen-free mice.

The effects of food restriction on immune function was investigated in germfree (GF) and specific pathogen-free (SPF) mice. They were maintained from five weeks of age under either full-fed or food-restricted conditions to 4.5 grams per day (equivalent to approximately 80% of full-fed intake) of a commercial diet. Longest survival rate was attained in food-restricted SPF mice followed by food-restricted GF, full-fed GF, and full-fed SPF animals. Food-restricted GF mice showed shorter survival rate than their SPF counterparts. This result suggests that food restriction may be just as effective as GF status for extending life span. Immune function declined significantly with age in full-fed groups of GF and SPF mice. In both food-restricted GF and SPF mice, mitogenic response to concanavalin A or lipopolysaccharide and antibody response to sheep red blood cells were lower early in life and became higher later in life as compared with full-fed mice. Hence, the maintenance of effective immunological function until old age may be the reason for food-restricted groups to live slightly longer than full-fed groups.     

Eliminating murine norovirus,Helicobacter hepaticus,and intestinal protozoa by embryo transfer for an entire mouse barrier facility

Pathogens can affect physiological and immunological reactions in immunocompromised animals and genetically engineered mice. Specifically, murine norovirus (MNV), Helicobacter, and intestinal protozoa are prevalent in rodent laboratory facilities worldwide. In this study, microbiological test results of the soiled bedding of sentinel mice showed the prevalence of MNV (50.9%, 28/55), Helicobacter hepaticus (29.1%, 16/55), Trichomonas spp. (14.5%, 8/55), and Entamoeba spp. (32.7%, 18/55). No single infections were detected as all cases were confirmed to have complex infections with two or four pathogens. In previous studies, the success rate of the cross-fostering method was not perfect; therefore, in this study, the entire mouse strain of the SPF rodent facility was rederived using embryo transfer. For up to three years, we confirmed that the results were negative with regular health surveillance tests. Embryo transfer was, thus, determined to be an effective method for the rederivation of specific pathogen free (SPF) barrier mouse facilities. This is the report for the effectiveness of embryo transfer as an example of successful microbiological clean-up of a mouse colony with multiple infections in an entire SPF mouse facility and embryo transfer may be useful for rederiving.     

Trichinella spiralis: enteric mucin-related response to experimental infection in conventional and SPF pigs

Duodenal and jejunal responses to infection with Trichinella spiralis were compared in weaned piglets with a "normal dirty" vs. a "clean SPF" gut flora. Histochemical staining of neutral, acidic, sialylated, and sulphated residues was used to assess biosynthetic responses in mucin-secreting goblet cells. Peanut and Ulex lectins were also used to assess responses within the intestinal glycocalyx. Histomorphometric analysis was undertaken to evaluate the distribution and staining patterns of goblet cells in villi and crypts. Our analysis showed that stored mucin within goblet cells increased more in the infected conventional animals than in the infected SPF group. This was accompanied by changes in the pattern of sulphation and sialylation in the duodenum and jejunum. The thickness of the glycocalyx was increased in both duodenum and jejunum in both infected groups. However, this effect was greater for the infected SPF animals than the infected conventional animals. No significant differences were observed between uninfected conventional and uninfected SPF pigs.     

A survey of Pneumocystis carinii infection in research mouse colonies in Japan.

To determine the frequency of Pneumocystis carinii infection in mouse colonies maintained for biomedical research in medical colleges or medical faculties in universities in Japan, 409 nu/nu mice were sent to 43 animal facilities from a P. carinii-free colony. The animals were housed for 6 months in groups of 3 to 10 animals per room, and examined for the presence of parasites and infection. Colonies in 10 (24.4%) of 41 facilities were positive for the infection. Of 383 animals in 69 rooms, the organism was detected in 66 (17.2%) animals in 13 (18.8%) rooms. The difference in the proportion of rooms where mice were positive for P. carinii is clearly seen among these three groups; SPF mouse rooms (4 of 38 rooms, 10.5%), SPF mouse rooms with breeding units (5 of 25 rooms, 20.0%) and conventional mouse rooms (4 of 6 rooms, 66.7%). The survey indicates that strict housing arrangements and husbandry techniques are necessary to keep SPF mice free from P. carinii infection.     

The use of biotyping of Enterobacteriaceae as an evaluation of the isolation efficacy of laboratory animals.

2 sets of data are given of biotyping the Enterobacteriaceae from the faecal flora of SPF rats and cats housed with different isolation arrangements. The method is presented as an evaluation of the isolation efficiency of laboratory animals. Biotypes which were determined on almost every occasion belonged to the resident flora of the animals while those found occasionally represented contamination. Contaminating bacteria are cleared from an SPF breeding unit by colonization resistance and the continuous efflux of animals from the unit. A parameter of isolation efficacy is the number of different biotypes determined in time. With optimal isolation arrangements a stable bacterial flora of permanently colonized biotypes is obtained in the animals.     

Immunoregulation mediated by the sympathetic nervous system,II

The effect of acute immunization or continuous environmental antigenic exposure on nor-adrenaline (NA) content of rat lymphoid organs was studied. Immunization with sheep red blood cells resulted in a decrease in splenic NA content. In other experiments, NA levels were determined in lymphoid and nonlymphoid organs of SPF rats exposed to naturally occurring environmental antigens, as well as in germ-free animals of the same strain. Spleen, thymus, and lymph nodes in SPF rats contained about 50% less NA than germ-free animals. With the exception of the adrenals, nonlymphoid organs showed no such a difference. Taken as a whole, the present results and our previous data on immunosuppressive influences of the sympathetic innervation on lymphoid organs support the notion that the sympathetic system plays a role in immunoregulation.     

S-phase fraction determined on fine needle aspirates is an independent prognostic factor in breast cancer – a multivariate study of 770 patients

Background: The prognostic significance of DNA ploidy and the S-phase fraction (SPF) have been extensively studied in breast cancer, but their clinical utility remains controversial. The type of tumour material can substantially influence flow cytometric DNA measurements. Material obtained by fine needle aspiration (FNA) biopsy is very suitable for flow cytometric DNA analysis because it contains a low proportion of non-tumour cells and less debris than tissue samples. Methods: The prognostic significance of DNA ploidy and SPF, determined on FNA samples, was analysed in 770 breast cancer patients, diagnosed between 1992 and 1997. DNA ploidy and SPF were determined at the time of diagnosis as part of the diagnostic work-up. The median follow-up was 90 months. Survival analysis included overall cancer specific survival (OS), disease free survival (DFS) and survival after recurrence (SAR). Other variables included in survival analyses were age, histological grade, histological type, lymph node status and tumour size. Disease free interval and the site of recurrence were also included in SAR analysis. Results: DNA ploidy and SPF correlated with tumour type, size, lymph node involvement and, especially, tumour grade. In a univariate analysis, both aneuploidy and high SPF were associated with shorter OS, DFS and SAR, but only SPF retained its independent prognostic significance in multivariate analyses. Independent prognostic variables for OS were node status, histological grade, SPF and tumour size. Node status, histological grade and SPF were independent predictors of DFS, while the site of recurrence, SPF, histological grade, disease free interval and age were independent predictors of SAR. Conclusions: DNA ploidy and SPF can be efficiently and routinely determined on FNA samples. High SPF is independently associated with a worse clinical outcome of patients with breast cancer. Although SPF and histological grade share prognostic information to some degree, SPF provides additional, less subjective prognostic information. The prognostic value of SPF determined on FNA samples could be even more relevant in neoadjuvant settings and for patients not amenable for surgical treatment, when histological grade cannot be assessed.     

Establishing a campylobacter-free pig population through a top-down approach

Fattening pigs are often infected with campylobacter. To eliminate campylobacter from the pig population, a top-down approach, involving the breeding and reproduction farms, seems appropriate. In order to investigate the effectiveness of a top-down approach, sows' faeces from the following farms were analysed for the presence of campylobacter: one specific pathogen free (SPF) farm, three top-breeding farms with no connection with SPF breeding, and a breeding farm repopulated with SPF sows after a period of vacancy (farm 5). The faeces samples from the SPF farm were free from campylobacter. The three top-breeding farms provided faeces samples which were 98% positive for campylobacter. However, only 22% of the faeces samples from farm 5 were positive for campylobacter. In a period of 20 months, the percentage of sows infected with campylobacter on farm 5 did not significantly increase. Genetic typing with ERIC-PCR and RFLP of campylobacter isolates from one of the top-breeding farms and from farm 5 showed a high diversity of campylobacter types. The results suggest that a campylobacter-free pig population can be established in breeding farms by combining a top-down approach (campylobacter-free top-breeding farms) with a strict regime of hygiene management.     

SNP‐based genetic characterization of the Tulane National Primate Research Center's conventional and specific pathogen‐free rhesus macaque (Macaca mulatta) populations

Background

The rhesus macaque is an important biomedical model organism, and the Tulane National Primate Research Center (TNPRC) has one of the largest rhesus macaque breeding colonies in the United States.

Methods

SNP profiles from 3266 rhesus macaques were used to examine the TNPRC colony genetic composition over time and across conventional or SPF animals of Chinese and Indian ancestry.

Results

Chinese origin animals were the least genetically diverse and the most inbred; however, since their derivation from their conventional forebearers, neither the Chinese nor the Indian SPF animals exhibit any significant loss of genetic diversity or differentiation.

Conclusions

The TNPRC colony managers have successfully minimized loss in genetic variation across generations. Although founder effects and bottlenecks among the Indian animals have been successfully curtailed, the Chinese subpopulation still show some influences from these events.     

医学实验动物屏障环境内独立通气笼具的安放

目的探讨屏障系统内安放独立通气笼盒（Individually Ventilated Cages,IVC）双保险模式饲养实验啮齿类动物,力求动物饲养、实验的全过程达到SPF级;降低动物实验室硬件建造费和维持费（节能）。方法通过改建一间屏障环境动物实验室,对空气洁净度等相关项目数据按GB14925-2001方法检定,并在该实验室内安放SPC级IVC饲养动物;在超净工作台内做打针投药等动物实验。结果改建的屏障环境动物实验室和IVC,各项检测数据均达到新国标要求。结论医学实验动物屏障环境内安放IVC饲养啮齿类动物、做动物实验,这种双保险模式能全过程达到SPF级要求;饲养、实验人员操作较简便,节能。     

Higher resistance of germfree mice to dianhydrodulcitol, a lymphotropic cytostatic agent

The same dose of dianhydrodulcitol (DAD) produced a lower mortality rate among germfree mice than among SPF or conventional C3H mice. On the other hand, it caused graver lymphoid atrophy in germfree mice. Their higher resistance, as evidenced by the mortality rate, can be explained on the basis of a histological study of the ileum. It showed milder alterations of the intestinal wall in germfree than in SPF mice. The lymphotropic cytostatic agent had a less direct toxic effect in germfree mice, due to the lacking damaging effect of endotoxin from the normal intestinal flora.     

Intestinal flora of animal models of human diseases as an environmental factor

Genetically-engineered animals are known to be useful in clarifying the functions of many genes and as animal models for human diseases. However, it has been widely reported that pathophysiology is not expressed in these animals when they become germfree or SPF animals, i.e., the pathophysiology is not the result of genes alone and a combination of gene function and intestinal flora as an environmental factor are necessary. It is important to determine the roles of each of these two factors by pathophysiological analysis. Gnotobiotic mice were produced by establishment of specified bacterial species in germfree animals to form the intestinal flora of SPF animals and they were placed in barrier facilities. Measures have been taken against infections by bacteria such as Pseudomonas aeruginosa and Enterobacter cloacae. In addition, gnotobiotic mice with a highly normal physiology are required. Analysis of the effects of each bacterial species and combinations of bacteria on in vivo functions, i.e., the cross-talk between the host and intestinal flora, is essential in the creation of better laboratory animals. Monitoring of the intestinal flora, a key factor in the colonies produced, is a topic for future research.     

Percutaneous osseointegrated prostheses for amputees: Limb compensation in a 12-month ovine model

Percutaneous osseointegrated prostheses are being investigated as an alternative strategy to attach prosthetic limbs to patients. Although the use of these implants has shown to be promising in clinical trials, the ability to maintain a skin seal around an osseointegrated implant interface is a major challenge to prevent superficial and deep periprosthetic infections. The specific aim of this study was to establish a translational load-bearing ovine model to assess postoperative limb compensation and gait symmetry following a percutaneous osseointegrated implant. We tested the following hypotheses: (1) the animals would return to pre-amputation limb loads within 12-months; (2) the animals would return to a symmetrical gait pattern (stride length and time in stance) within 12-months. The results demonstrated that one month following surgery, the sheep loaded their amputated limb to a mean value of nearly 80% of their pre-amputation loading condition; by 12-months, this mean had dropped to approximately 74%. There was no statistical differences between the symmetry of the amputated forelimb and the contralateral forelimb at any time point for the animals stride length or the time spent in the stance phase of their gait cycle. Thus, the data showed that while the animals maintained symmetric gait patterns, they did not return to full weight-bearing after 12-months. The results of this study showed that a large animal load-bearing model had a symmetric gait and was weight bearing for up to 12 months. While the current investigation utilizes an ovine model, the data show that osseointegrated implant technology with postoperative follow-up can help our human patients return to symmetric gait and maintain an active lifestyle, leading to an improvement in their quality of life following amputation.