Specific Pathogen-free Laboratory Research Articles

Reduced T Cell Priming in Microbially Experienced "Dirty" Mice Results from Limited IL-27 Production by XCR1+ Dendritic Cells.

Successful vaccination strategies offer the potential for lifelong immunity against infectious diseases and cancer. There has been increased attention regarding the limited translation of some preclinical findings generated using specific pathogen-free (SPF) laboratory mice to humans. One potential reason for the difference between preclinical and clinical findings lies in maturation status of the immune system at the time of challenge. In this study, we used a "dirty" mouse model, where SPF laboratory mice were cohoused (CoH) with pet store mice to permit microbe transfer and immune system maturation, to investigate the priming of a naive T cell response after vaccination with a peptide subunit mixed with polyinosinic-polycytidylic acid and agonistic anti-CD40 mAb. Although this vaccination platform induced robust antitumor immunity in SPF mice, it failed to do so in microbially experienced CoH mice. Subsequent investigation revealed that despite similar numbers of Ag-specific naive CD4 and CD8 T cell precursors, the expansion, differentiation, and recall responses of these CD4 and CD8 T cell populations in CoH mice were significantly reduced compared with SPF mice after vaccination. Evaluation of the dendritic cell compartment revealed reduced IL-27p28 expression by XCR1+ dendritic cells from CoH mice after vaccination, correlating with reduced T cell expansion. Importantly, administration of recombinant IL-27:EBI3 complex to CoH mice shortly after vaccination significantly boosted Ag-specific CD8 and CD4 T cell expansion, further implicating the defect to be T cell extrinsic. Collectively, our data show the potential limitation of exclusive use of SPF mice when testing vaccine efficacy.

Reduced T cell priming to vaccination in microbially-experienced (‘dirty’) mice

Abstract Successful vaccination strategies can provide lifelong immunity against infectious diseases and cancer. Vaccines are widely considered one of the greatest advancements in public health; however, recent failures in vaccine development indicate our inadequate understanding of the immune response to vaccination. There has been increased attention recently regarding the limited translation of preclinical findings generated using specific pathogen free (SPF) laboratory mice to humans. One potential reason for the difference between laboratory mice and humans lies in maturation status of the immune system at the time of challenge. We have been using a ‘dirty’ mouse model, where SPF laboratory mice are cohoused with pet store mice to permit pathogen transfer and immune system maturation, to investigate the naive T cell response after peptide vaccination with Trivax (polyI:C and anti-CD40 mAb) or Complete Freund’s Adjuvant (CFA). Despite having no differences in the number of antigen-specific naïve CD4 and CD8 T cell precursors, the expansion of these CD4 and CD8 T cell populations in cohoused (CoH) B6 mice was significantly reduced (compared to SPF B6 mice) following vaccination with either Trivax or CFA. Furthermore, memory recall responses to cognate antigen – as measured by proliferative capacity or cytokine production – were reduced in the CoH mice. Evaluation of the dendritic cell compartment revealed reduced expression of costimulatory molecules (e.g., CD80/CD86) on DC from CoH mice shortly after vaccination. One consequence of the muted T cell priming in CoH mice was reduced antitumor immunity after vaccination. Collectively, our data show the potential limitation of exclusive use of SPF mice when testing vaccine efficacy.

Study of MAIT Cell Activation in Viral Infections In Vivo.

MAIT cells are abundant, highly evolutionarily conserved innate-like lymphocytes expressing a semi-invariant T cell receptor (TCR), which recognizes microbially derived small intermediate molecules from the riboflavin biosynthetic pathway. However, in addition to their TCR-mediated functions they can also be activated in a TCR-independent manner via cytokines including IL-12, -15, -18, and type I interferon. Emerging data suggest that they are expanded and activated by a range of viral infections, and significantly that they can contribute to a protective anti-viral response. Here we describe methods used to investigate these anti-viral functions in vivo in murine models. To overcome the technical challenge that MAIT cells are rare in specific pathogen-free laboratory mice, we describe how pulmonary MAIT cells can be expanded using intranasal bacterial infection or a combination of synthetic MAIT cell antigen and TLR agonists. We also describe protocols for adoptive transfer of MAIT cells, methods for lung homogenization for plaque assays, and surface and intracellular cytokine staining to determine MAIT cell activation.

Prevalence of lapine rotavirus, astrovirus, and hepatitis E virus in Canadian domestic rabbit populations

Lapine rotavirus and astrovirus have been associated with disease in rabbits, and there is strong evidence of zoonotic transmission of lapine hepatitis E virus (HEV). Outbreaks of enteritis are common on commercial meat farms, resulting in poor welfare, high rabbit mortality, and significant financial losses for rabbit producers. Currently, none of these viruses are routinely tested by diagnostic laboratories. In this study, we assessed the prevalence of rotavirus, astrovirus, and HEV RNA in 205 pooled and individual fecal samples from healthy Canadian laboratory, companion, shelter and commercial meat rabbit populations. Viral RNA were extracted and amplified via RT-PCR using virus-specific primers. Positive samples from the first cohort of samples tested were sequenced and aligned to previously identified viruses to confirm the products. Almost 45% (13/29) of the surveyed commercial rabbit farms were astrovirus-positive. Three commercial meat rabbit samples were positive for rotavirus, and either astrovirus or HEV RNA was also detected. Three companion rabbit samples also tested positive for lapine HEV. Samples from specific pathogen-free laboratory animals were negative for all viruses. Sequencing results showed highest identity to rotavirus A strain 30–96, lapine astrovirus strain 2208 and lapine HEV strain CMC-1. These results permit a better understanding of the prevalence of rotavirus, astrovirus, and hepatitis E virus in Canadian domestic rabbit populations, and continued screening for viruses may help to reduce risk of zoonotic agent transmission as well as providing a better understanding of potential causative agents of rabbit enteritis.

Detection of Immunodominant Proteins of Felis catus Gammaherpesvirus 1

We recently identified and sequenced a novel herpesvirus of domestic cats, Felis catus gammaherpesvirus 1 (FcaGHV1). FcaGHV1 is a member of the gammaherpesvirus subfamily which also includes the human cancer-associated herpesviruses, Epstein-Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV). As a first step toward developing a serologic assay to detect exposure to FcaGHV1, we are seeking to determine which viral proteins elicit an antibody response in naturally occurring domestic cat infections. We cloned selected FcaGHV1 genes into a mammalian expression vector and performed transfections of a feline cell line for expression of recombinant FcaGHV1 proteins. We fixed cells with paraformaldehyde and methanol-acetone and tested reactivity to serum from cats naturally infected with FcaGHV1 using immunofluorescent antibody staining. An FIV immunoflouresence test was developed as a positive control for transfection and assay function. Serum from specific pathogen-free laboratory cats served as negative controls. Preliminary data from 9 cats with FcaGHV1 infection indicates that capsid protein ORF 65 and tegument protein ORF38 may elicit antibodies during naturally occurring FcaGHV1 infection. Results of this study will suggest which FcaGHV1 proteins are immunodominant during natural infection. With this information we plan to develop a serologic assay and further evaluate FcaGHV1 as a model for EBV and KSHV.

Three Weeks of Anti-orthostatic Suspension had no Effect on Immune Status in a Specific-Pathogen-Free Enfvironment

Anti-Orthostatic Suspension (AOS) is a well-known murine ground-based model of simulated microgravity. However, because no commercial equipment is available for AOS in Specific-Pathogen-Free (SPF) laboratories no previous study has been conducted to examine the effect of AOS on allergic immunity. Accordingly, we developed an AOS cage suitable for SPF conditions, and evaluate its reliability and the effect of 3 weeks of AOS on immunity in a mouse model. An AOS cage were developed using stainless steel components. Fourteen female BALB/c mice were allocated to Group A (control group, n=7) or Group B (AOS, n=7). Body weights and thickness of posterior thigh muscles were measured before and after 3 weeks of AOS, and serum IgE and the titers of cytokines (IL-4, IL-5, IL-10, IL-13, and IFN- γ) in bronchoalveolar lavage (BAL) fluid were compared, as were lung histologic findings. The SPF condition was successfully maintained. No significant difference in weight gain was observed between groups A (0.9 ±1.0 g) and B (0.8 ±1.1 g, P>0.05) after the 3-week experimental period. The mean thickness of posterior thigh muscles in Group B was significantly lower than in Group A (0.7 ±0.2 versus -0.4 ±0.3 mm, P =0.001). However, group serum IgE titers, and IL-4, IL-5, IL-10, IL-13, and IFN- γ titers in BAL fluid were non-significantly different. No intergroup difference was found by histopathologic examinations of lungs. In conclusion, using AOS equipment in a SPF laboratory, immune status was not found to be significantly affected by 3 weeks AOS in healthy mice.

Hand-reared common swifts (Apus apus) in a wildlife rehabilitation centre: assessment of growth rates using different diets

Common swift (Apus apus) orphans represent an important number of admissions to wildlife rehabilitation centres in Europe. Rehabilitation centres may encounter difficulties in the hand-rearing of large numbers of insectivore chicks if they use commercially available insects, which are usually expensive and nutritionally incomplete. These constraints have created the necessity for alternative diets; however, these may not be optimal for hand-rearing purely insectivorous species. In this study, 116 orphan common swift nestlings were hand-reared during June and July 2008 and 2009 in the Torreferrusa Wildlife Rehabilitation Centre (Catalonia, northern Spain). We assessed growth rates and final fledgling weight under four different diets, comparing the results to those of wild parent-raised common swifts. Clinical condition at admission was the main variable predicted to influence the results. The four diets were (1) rat mince diet, a specific pathogen-free laboratory rat mince; (2) kibble diet, a formula based on a high-protein–low-carbohydrate cat food; (3) cricket diet, based on house crickets (Acheta domesticus) and wax moth larvae (Galleria mellonella); and (4) mealworm diet, based on mealworm larvae (Tenebrio molitor). Reference adult weights of wild animals were obtained from the literature (41.5g ± 2.42 SD). The results showed significant differences in final weights, which were considerably lower for hand-reared animals on the non-insect diets (rat mince diet: 32.8g ± 2.7; kibble diet: 32.5g ± 3.7). The final weights in both insect diet groups were satisfactory, with values close to those observed in the wild (cricket diet: 40.1g ± 4.0; mealworm diet: 40.3g ± 3.1). The results of this research highlight the need to implement changes in diet protocols when using non-insect-based diets.

Natural infection of murine norovirus in conventional and specific pathogen-free laboratory mice

Noroviruses cause most cases of acute viral gastroenteritis worldwide. The lack of a cell culture infection model for human norovirus necessitates the use of molecular methods and/or viral surrogate models amenable to cell culture to predict norovirus inactivation. Murine norovirus (MNV) may be used to construct a small animal model for studying the biology and pathogenesis of noroviruses because MNV is the only norovirus that replicates in cell culture and a small animal model. However, recent studies have shown that natural MNV infection is widespread in laboratory mouse colonies. We investigated MNV infection in both conventional and specific pathogen-free (SPF) genetically modified mice from Japan and the US, and commercial mice from several animal breeders in Japan, using serological and molecular techniques. MNV antibodies were detected in 67.3% of conventional mice and 39.1% of SPF mice from Japan and 62.5% of conventional mice from the US. MNV antibodies were also found in 20% of commercial SPF C57BL/6 mice from one of three breeders. Partial gene amplification of fecal isolates from infected animals showed that the isolates were homologous to reported MNV sequences. These results suggest that both conventional and SPF laboratory mice, including commercial mice, are widely infected with MNV, which might require considerable attention as an animal model of human disease.

Age-related dysregulation of CD8+ T cell memory specific for a persistent virus is independent of viral replication.

The immune system devotes substantial resources to the lifelong control of persistent pathogens, which were hypothesized to play an important role in immune aging. Specifically, the presence of latent herpesviruses has been correlated with immune exhaustion and shorter lifespan in octogenarians. But neither the causality nor the mechanistic link(s) were established, and the relative roles of persistent antigenic stimulation and of virus-independent homeostatic disturbances in T cell aging remain unresolved. We longitudinally analyzed expansion, contraction, and long-term maintenance of CD8(+) T cells responding to localized infection with a latent virus, HSV-1. Young mice exhibited the expected expansion and contraction of HSV-1-specific cells and the stable maintenance of memory T cells into advanced adulthood. However, upon entry into senescence, many (>40%) animals exhibited an accumulation in Ag-specific cells (memory inflation) which in some animals was comparable to that observed in acute infection. Inflation occurred to the same extent in control mice and mice continuously treated with the anti-HSV drug famciclovir, which inhibits viral replication and was able to reduce expression of the glycoprotein B. Age-related inflation was also found long after infection with an acute virus. The inflating cells largely maintained Ag-specific function, and exhibited typical central memory phenotype, with no signs of Ag-specific activation. They exhibited increased expression of CD122 and CD127, akin to the Ag-independent T cell clonal expansions found in old specific pathogen-free laboratory mice. This collectively suggests that, in this model, the inflating cells may be selected for high responsiveness to environmental cytokines largely in an Ag-independent manner.

Model of septic shock induced by live E. coli (O18) in a laboratory rat

This study was concerned with the development of induced septic shock in a laboratory rat using a series of measurements including body temperature, heart and respiratory rates, haematocrit value, red and white blood cell counts, differential leukocyte count, haemoglobin value, glycaemia, analysis of arterial blood gases, and serum levels of interleukin 6 (IL-6) during the first five hours. A total of 12 specific pathogen free (SPF) laboratory rats were used for the study. Septic shock was induced under general anaesthesia by introducing live <I>E. coli</I> (O18) into the jugular vein in the dose of 1 × 10<sup>9</sup> per 100 g of body weight (group SESH). Clinical measurements and blood collection from <I>a. carotis</I> were performed just prior to, and then 1.5 and 5 h after the administration of <I>E. coli.</I> The control group (C) contained 9 SPF laboratory rats which received physiological saline only, at the same volume into the jugular vein, and blood collection followed according to the same scheme as above described for group SESH. The results of the experiment showed that changes in clinical, haematological and biochemical parameters could be detected as early as 1.5 hours after induction. These changes correspond with the activation of an inflammatory reaction and the development of metabolic acidosis. They are accompanied by a considerable rise in IL-6 already 1.5 h after the application of live <I>E. coli</I> and after 5 h the levels exceeded 2 000 pg/ml in all experimental animals. Our results clearly document the importance of IL-6 for the early detection of developing septic shock and of some less specific but routinely determined parameters such as white blood cell count and base excess.

Molecular, Cellular, and Antigen Requirements for Development of Age-Associated T Cell Clonal Expansions In Vivo

T cell aging manifests itself both at the cellular (cell-autonomous defects in signaling) and at the population (age-related dysregulation of T cell homeostasis) levels. A prominent contributor to the latter is the appearance of T cell clonal expansions (TCE), with a potential to impair immune defense. In this study, we investigated molecular, cellular, and Ag requirements for TCE development. Of the mutant mice tested, old animals lacking MHC class I exhibited 7-fold fewer TCE than controls, with a 7-fold reduction in TCE. By contrast, animals lacking only one of the MHC class I molecules (Kb or Db), or IL-7R, or devoid of T cell renewal via adult thymectomy, all exhibited significant increases in TCE incidence. This increase directly correlated to lymphopenia, increased CD8 T cell turnover and an accumulation of memory-phenotype T cells. These data suggested that homeostatic cell division in the CD8 compartment enhances the formation of TCE. Repeated immunization with peptide/adjuvant did not result in an increase in Ag-specific TCE; however, adjuvant alone increased TCE incidence. In these experiments, therefore, nonspecific and/or homeostatic proliferation was more efficient in generating TCE in mice than repeated Ag-driven stimulation, suggesting that many, if not most, TCE in specific pathogen-free laboratory mice may be Ag-independent.

Microbiologically monitored fumigation of a newly built SPF laboratory rodent facility.

The initial sanitization and sterilization of a newly built animal facility for the breeding and holding of specific pathogen free (SPF) rats and mice is described. The fumigation programme was started with methyl bromide treatment directed primarily against arthropods, followed by ammonia spray to kill coccidial oocysts and concluded by three formaldehyde treatments with fog and spray against bacteria and viruses. The practicalities and problems involved are described in detail and the rationale and purpose of the programme and its monitoring are discussed. The report is expected to contribute towards the establishment of a rational, efficient and standardized fumigation programme for SPF animal facilities, under increasing constraints of safety and environmental considerations concerning pollution with toxic and corrosive agents.