Commensal oral microbiota induces osteoimmunomodulatory effects separate from systemic microbiome in mice.

Commensal microbes critically regulate skeletal homeostasis, yet the impact of specific microbiota communities on osteoimmune response mechanisms is unknown. To discern osteoimmunomodulatory effects imparted by the commensal oral microbiota that are distinct from the systemic microbiota, osteoimmunology studies were performed in both alveolar bone and nonoral skeletal sites of specific pathogen-free (SPF) versus germ-free (GF) mice and SPF mice subjected to saline versus chlorhexidine oral rinses. SPF versus GF mice had reduced cortical/trabecular bone and an enhanced pro-osteoclastic phenotype in alveolar bone. TLR signaling and Th17 cells that have known pro-osteoclastic actions were increased in alveolar BM, but not long BM, of SPF versus GF mice. MHC II antigen presentation genes and activated DCs and CD4+ T cells were elevated in alveolar BM, but not long BM, of SPF versus GF mice. These findings were substantiated by in vitro allostimulation studies demonstrating increased activated DCs derived from alveolar BM, but not long BM, of SPF versus GF mice. Chlorhexidine antiseptic rinse depleted the oral, but not gut, bacteriome in SPF mice. Findings from saline- versus chlorhexidine-treated SPF mice corroborated outcomes from SPF versus GF mice, which reveals that the commensal oral microbiota imparts osteoimmunomodulatory effects separate from the systemic microbiome.

New Insights into the Immune System Using Dirty Mice.

The mouse (Mus musculus) is the dominant organism used to investigate the mechanisms behind complex immunological responses because of their genetic similarity to humans and our ability to manipulate those genetics to understand downstream function. Indeed, our knowledge of immune system development, response to infection, and ways to therapeutically manipulate the immune response to combat disease were, in large part, delineated in the mouse. Despite the power of mouse-based immunology research, the translational efficacy of many new therapies from mouse to human is far from ideal. Recent data have highlighted how the naive, neonate-like immune system of specific pathogen-free mice differs dramatically in composition and function to mice living under barrier-free conditions (i.e., "dirty" mice). In this review, we discuss major findings to date and challenges faced when using dirty mice and specific areas of immunology research that may benefit from using animals with robust and varied microbial exposure.

Prevention of infection in xenotransplantation: Designated pathogen-free swine in the safety equation.

Post-transplantation infections are common. In immunosuppressed human xenograft recipients, infection is most likely to be due to the same pathogens seen in human allotransplantation. However, organisms derived from swine and transmitted with xenografts have the potential to cause novel infections in xenograft recipients. The specific organisms likely to cause infection or "xenosis" are unknown but are postulated to be like those causing infection in allograft recipients. On this basis, theoretical exclusion criteria have been developed to guide the development of source animal herds. Herds developed based on the exclusion of potential human pathogens have been termed "designated pathogen-free" (DPF). Lists of potential pathogens will require revision with changing epidemiology of infection in swine worldwide and clinical experience. Development of new microbiological assays is required both for animal screening and in clinical diagnosis should infections occur. Genetic modifications of swine have the potential to eliminate certain infectious agents such as the porcine endogenous retrovirus; infectious complications of such modifications have not been observed. Unexpected, off target effects of genetic modifications require further study. Monitoring for infection in asymptomatic recipients is important to define infectious risks which are unknown in the absence of clinical trials data. Advanced microbiological techniques may be applied to diagnose and prevent infection in xenograft recipients.

Response to mechanical loading in rat Achilles tendon healing is influenced by the microbiome.

We have previously shown that changes in the microbiome influence how the healing tendon responds to different treatments. The aim of this study was to investigate if changes in the microbiome influence the response to mechanical loading during tendon healing. 90 Sprague-Dawley rats were used. Specific Opportunist and Pathogen Free (SOPF) rats were co-housed with Specific Pathogen Free (SPF) rats, carrying Staphylococcus aureus and other opportunistic microbes. After 6 weeks of co-housing, the SOPF rats were contaminated which was confirmed by Staphylococcus aureus growth. Clean SOPF rats were used as controls. The rats were randomized to full loading or partial unloading by Botox injections in their calf muscles followed by complete Achilles tendon transection. Eight days later, the healing tendons were tested mechanically. The results were analysed by a 2-way ANOVA with interaction between loading and contamination on peak force as the primary outcome and there was an interaction for both peak force (p = 0.049) and stiffness (p = 0.033). Furthermore, partial unloading had a profound effect on most outcome variables. In conclusion, the response to mechanical loading during tendon healing is influenced by changes in the microbiome. Studies aiming for clinical relevance should therefore consider the microbiome of laboratory animals.

Emerging Insights on the Interaction Between Anticancer and Immunosuppressant Drugs and Intestinal Microbiota in Pediatric Patients.

Diseases affecting the immune system, such as inflammatory bowel disease (IBD), juvenile idiopathic arthritis (JIA), and acute lymphoblastic leukemia (ALL), are pathological conditions affecting the pediatric population and are often associated with alterations in the intestinal microbiota, such as a decrease in bacterial diversity. Growing evidence suggests that gut microbiota can interfere with chemotherapeutic and immunosuppressant drugs, used in the treatment of these diseases, reducing or facilitating drug efficacy. In particular, the effect of intestinal microflora through translocation, immunomodulation, metabolism, enzymatic degradation, and reduction of bacterial diversity seems to be one of the reasons of interindividual variability in the therapeutic response. Although the extent of the role of intestinal microflora in chemotherapy and immunosuppression remains still unresolved, current evidence on bacterial compositional shifts will be taken in consideration together with clinical response to drugs for a better and personalized therapy. This review is focused on the effect of the intestinal microbiota on the efficacy of pharmacological therapy of agents used to treat IBD, JIA, and ALL.

A Ligated Intestinal Loop Model in Anesthetized Specific Pathogen Free Chickens to Study Clostridium Perfringens Virulence.

Necrotic enteritis was studied in chickens using various in vivo infection models. Most of these use a combination of predisposing factors, such as coccidiosis and diet, with gavage or administration via the feed using Clostridium perfringens. In these models, the comparison of multiple C. perfringens strains for virulence studies requires a large number of hosts to obtain significant results. Mortality during the course of the study can be high depending on the experimental model, hence raising ethical concerns regarding animal welfare in research. The development of new infection models requiring fewer animals to study pathogenesis, yet providing statistically significant and valid results, is important in reducing animal use in research. Intestinal ligated loop models have been used to study clostridial infections in various species such as mice, rabbits and calves. Following surgical procedures to create ligated loop segments, C. perfringens strains are injected directly into the loops to establish a close contact between the bacteria and the intestinal mucosa. Samples of the small intestine and luminal contents are taken at the termination of the procedures after a few hours. Multiple bacterial strains can be inoculated in each animal, hence reducing the number of required subjects in the experiments. Also, procedures are performed under general anesthesia to reduce animal pain. In chickens, this model would be more appropriate than oral administration to compare C. perfringens strain pathogenicity because fewer animals are needed, no predisposing factors are required to induce the disease, and pain is controlled by analgesics. The intestinal ligated loop model is poorly described in chickens and standardization is essential for its optimal use. This manuscript provides all the necessary steps to create numerous intestinal ligated loops in chickens and brings information on the critical points to obtain valid results.

Early cellular responses of germ-free and specific-pathogen-free mice to Francisella tularensis infection.

Bacteria that are highly virulent, expressing high infectivity, and able to survive nebulization, pose great risk to the human population. One of these is Francisella tularensis, the etiological agent of tularemia. F. tularensis is a subject of intense scientific interest due to the fact that vaccines for its immunoprophylaxis in humans are not yet routinely available. One of the substantial obstacles in developing such vaccines is our insufficient knowledge of processes that initiate and regulate the expression of effective protective immunity against intracellular bacteria. Here, we present data documenting the different pattern of cellular behavior occurring in an environment unaffected by microbiota using the model of germ-free mice mono-associated with F. tularensis subsp. holarctica strain LVS in comparison with a classic specific-pathogen-free murine model during early stages of infection.

Salmonella Enteritidis with double deletion in phoP fliC and a competitive exclusion culture elicit substantial additive protective effects against Salmonella exposure in newly hatched chicks.

A live Salmonella Enteritidis vaccine (SE147N ΔphoP fliC), able to express both a homologous intestinal colonisation-inhibition effect and a systemic invasion-inhibition effect, was tested for its potential to generate a postulated additive protective effect in case of combined application with a competitive exclusion (CE) culture against Salmonella exposure in very young chicks. Both, SE147N ΔphoP fliC and the CE culture alone were highly protective against systemic and intestinal colonisation of the challenge strain in case of moderate Salmonella exposure, consequently, additive protective effects in combined use could not be detected. However, in case of high Salmonella Enteritidis challenge with 106 cfu/bird at day 3 of life the combination of the ΔphoP fliC vaccine and the CE culture resulted in a protective effect much more pronounced than either of the single preparations and most substantial compared to untreated control birds. The term additive protective effects reflects the recognition that exclusion effects by gut flora cultures and inhibition effects by Salmonella vaccines are caused by different mechanisms.

Innate immune responses induced by the saponin adjuvant Matrix-M in specific pathogen free pigs.

Saponin-based adjuvants have been widely used to enhance humoral and cellular immune responses in many species, but their mode of action is not fully understood. A characterization of the porcine transcriptional response to Matrix-M was performed in vitro using lymphocytes, monocytes or monocyte-derived dendritic cells (MoDCs) and in vivo. The effect of Matrix-M was also evaluated in specific pathogen free (SPF) pigs exposed to conventionally reared pigs. The pro-inflammatory cytokine genes IL1B and CXCL8 were up-regulated in monocytes and lymphocytes after Matrix-M exposure. Matrix-M also induced IL12B, IL17A and IFNG in lymphocytes and IFN-α gene expression in MoDCs. Several genes were indicated as up-regulated by Matrix-M in blood 18 h after injection, of which the genes for IFN-α and TLR2 could be statistically confirmed. Respiratory disease developed in all SPF pigs mixed with conventional pigs within 1-3 days. Two out of four SPF pigs injected with saline prior to contact exposure displayed systemic symptoms that was not recorded for the four pigs administered Matrix-M. Granulocyte counts, serum amyloid A levels and transcription of IL18 and TLR2 coincided with disease progression in the pigs. These results support further evaluation of Matrix-M as a possible enhancer of innate immune responses during critical moments in pig management.

Immunomodulatory and Anti-IBDV Activities of the Polysaccharide AEX from Coccomyxa gloeobotrydiformis.

A number of polysaccharides have been reported to show immunomodulatory and antiviral activities against various animal viruses. AEX is a polysaccharide extracted from the green algae, Coccomyxa gloeobotrydiformis. The aim of this study was to examine the function of AEX in regulating the immune response in chickens and its capacity to inhibit the infectious bursal disease virus (IBDV), to gain an understanding of its immunomodulatory and antiviral ability. Here, preliminary immunological tests in vitro showed that the polysaccharide AEX can activate the chicken peripheral blood molecular cells' (PBMCs) response by inducing the production of cytokines and NO, promote extracellular antigen presentation but negatively regulate intracellular antigen presentation in chicken splenic lymphocytes, and promote the proliferation of splenic lymphocytes and DT40 cells. An antiviral analysis showed that AEX repressed IBDV replication by the deactivation of viral particles or by interfering with adsorption in vitro and reduced the IBDV viral titer in the chicken bursa of Fabricius. Finally, in this study, when AEX was used as an adjuvant for the IBDV vaccine, specific anti-IBDV antibody (IgY, IgM, and IgA) titers were significantly decreased. These results indicate that the polysaccharide AEX may be a potential alternative approach for anti-IBDV therapy and an immunomodulator for the poultry industry. However, more experimentation is needed to find suitable conditions for it to be used as an adjuvant for the IBDV vaccine.

In situ hybridization to detect and localize signature cytokines of T-helper (Th) 1 and Th2 immune responses in chicken tissues.

The avian immune system has been shown to possess a repertoire of cytokines directing T-helper (Th) 1 and Th2 types of immune responses similar to that in mammals. The objective of this study was to establish in situ hybridization (ISH) for the localization of mRNA of selected signal cytokines, chicken interferon-γ (ChIFN-γ), chicken interleukin (ChIL)-4 and ChIL-13 in fixed tissues. RNA probes were generated to hybridize to 488, 318, and 417bp of the respective target mRNA. Probe concentrations ranging from 100ng/ml to 400ng/ml were shown to be suitable to label cells that expressed these cytokines. The specificity of every probe was verified using the respective sense probe. ChIFN-γ, ChIL-4 and ChIL-13 positive cells were observed in the lymphocytic infiltrations of liver and in the periarteriolar lymphatic sheaths of spleen collected from specific-pathogen-free chickens. ISH of these cytokines in a severely inflamed liver due to infiltration with the parasite Histomonas meleagridis revealed the expression of both ChIFN-γ and ChIL-13 mRNA in the mononuclear infiltrates. In conclusion, ChIFN-γ, ChIL-4 and ChIL-13 mRNA were efficiently localized by ISH, which supplies a valid technique to characterize immune responses in fixed tissues.

Head-to-head comparison of protocol modifications for the generation of collagen-induced arthritis in a specific-pathogen free facility using DBA/1 mice.

Collagen-induced arthritis (CIA) is a widely used mouse model for studying inflammatory arthritis (IA). However, CIA induction protocols differ between laboratories, and direct comparison between protocol variations has not been reported. To address this issue, DBA/1 mice housed in conventional and specific-pathogen free (SPF) facilities were administered various combinations of two doses of collagen type II (CII) in complete (CFA) or incomplete Freund's adjuvant (IFA); some mice were also injected with lipopolysaccharide (LPS) and/or additional CII at specific intervals. Mice were evaluated for IA over the subsequent 2 months. Depending directly on the combination of CII, CFA, IFA, and LPS used, the incidence of IA ranged between 20%-100%, and severity extended from mild to severe even in an SPF environment. Our results demonstrate for the first time in head-to-head comparisons that specific variations in the use of CII, CFA, IFA, and LPS can induce a range of arthritic disease intensity and severity in an SPF facility. Thus, distinct experimental settings can be designed for robust assessment of factors that either exacerbate or inhibit arthritis pathogenesis. Furthermore, by achieving 100% incidence in an SPF facility, the protocols provide a practical and humane benefit by reducing the number of mice necessary for experimental assessment.

Quantification of rHVT-F genome load in feather follicles by specific real-time qPCR as an indicator of NDV-specific humoral immunity induced by day-old vaccination in SPF chickens.

The purpose of this study was to look for a reliable molecular method for confirmation of uptake of recombinant turkey herpesvirus vaccine against Newcastle disease (rHVT-F) and for use as a valuable prediction tool of Newcastle disease virus (NDV)-specific immune response in chickens deprived of maternally derived antibody (MDA). A quantitative real-time polymerase chain reaction (real-time qPCR) specific to rHVT-F was developed. The method was applied to various tissue samples taken from specific pathogen free (SPF) chickens experimentally inoculated at day-old with one dose of rHVT-F vaccine over a 6-week period. Among the tested tissues, the rHVT-F vaccine was detected predominantly in the bursa of Fabricius (BF) and the lung for the first week, followed by a progressive decline from 9 days onwards. Then, an increase of genome load was observed in the feather follicles (FF) with a peak at 2 weeks, rising to a level almost 10(3)-fold greater than in the other tissues. Importantly, the rHVT-F genome load in FF appeared to be strongly correlated to the humoral immunity specific to NDV as evaluated by haemagglutination inhibition (HI) test and NDV-specific IgG, IgM and IgA ELISAs. This is the first report of quantification of rHVT-F vaccine in FF and its correlation with the induction of ND-specific immune response in chickens with no MDA. Our data indicate that the application of this real-time qPCR assay on FF samples taken from chickens in the field may be used to confirm rHVT-F vaccine administration and uptake with the important added benefit of offering a non-disruptive sampling procedure.

Recombinant feline coronaviruses as vaccine candidates confer protection in SPF but not in conventional cats.

Feline infectious peritonitis virus (FIPV) is a major pathogen of Felidae. Despite the extensive efforts taken in the past decades, development of the "ideal" live attenuated FIPV vaccine was not successful yet. In the present study, we provide data of immunisation experiments with a recombinant FCoV pair differing only in the truncation (PBFIPV-DF-2) or completion (PBFIPV-DF-2-R3i) of their ORF3abc regions. In our previous in vivo studies, these viruses proved to show the characters of low virulent or avirulent FCoV phenotypes, respectively. Therefore, we hypothesised the ability of these viruses, as possible vaccine candidates, in conferring protection in specific pathogen free (SPF) Domestic Shorthair as well as in conventional purebred British Shorthair cats. In SPF cats, after two oronasal and two intramuscular vaccinations with two weeks intervals, both vaccine candidates provided 100% protection against lethal homologous challenge with the highly virulent FIPV DF-2 strain. In contrast, the conventional purebred British Shorthair cats did not develop protection when they were immunised with the same vaccination regimes. In these groups 100% of the PBFIPV-DF-2-R3i immunised animals developed antibody-dependent enhancement (ADE). Prolonged survival was observed in 40% of the animals, while 60% showed fulminant disease course. Genetic and more probably immunological differences between the SPF and non-SPF purebred kittens can explain the different outcome of the vaccination experiment. Our data highlight the diverse immune responses between SPF and conventional cats and suggest a decisive role of previous infection by heterologous causative agents in the outcome of the vaccination against FIP.

Management of hepatitis E virus (HEV) zoonotic transmission: protection of rabbits against HEV challenge following immunization with HEV 239 vaccine.

Hepatitis E virus (HEV) constitutes a significant health burden worldwide, with an estimated approximately 33% of the world's population exposed to the pathogen. The recent licensed HEV 239 vaccine in China showed excellent protective efficacy against HEV of genotypes 1 and 4 in the general population and pregnant women. Because hepatitis E is a zoonosis, it is also necessary to ascertain whether this vaccine can serve to manage animal sources of human HEV infection. To test the efficacy of the HEV 239 vaccine in protecting animal reservoirs of HEV against HEV infection, twelve specific-pathogen-free (SPF) rabbits were divided randomly into two groups of 6 animals and inoculated intramuscularly with HEV 239 and placebo (PBS). All animals were challenged intravenously with swine HEV of genotype 4 or rabbit HEV seven weeks after the initial immunization. The course of infection was monitored for 10 weeks by serum ALT levels, duration of viremia and fecal virus excretion and HEV antibody responses. All rabbits immunized with HEV 239 developed high titers of anti-HEV and no signs of HEV infection were observed throughout the experiment, while rabbits inoculated with PBS developed viral hepatitis following challenge, with liver enzyme elevations, viremia, and fecal virus shedding. Our data indicated that the HEV 239 vaccine is highly immunogenic for rabbits and that it can completely protect rabbits against homologous and heterologous HEV infections. These findings could facilitate the prevention of food-borne sporadic HEV infection in both developing and industrialized countries.

Pathogen-free husbandry conditions alleviate behavioral deficits and neurodegeneration in AD10 anti-NGF mice.

It has been suggested that systemic infection, occurring during aging and chronic neurodegenerative diseases, can evoke an immune response that aggravates the progression of neurodegeneration and cognitive decline. It has been shown that the AD11 neurodegeneration mouse model, expressing a recombinant anti-nerve growth factor (NGF) antibody, shows a milder phenotype when housed in murine pathogen-free (MPF) conditions with respect to AD11 mice reared in conventional (CV) housing. AD10 mice, a variant of the anti-NGF AD11 model, expressing only an immunoglobulin light chain for the transgenic anti-NGF antibody, in the absence of the corresponding transgenic antibody chain VH, exhibit a complex neurodegenerative phenotype, similar to that of AD11 mice. Here we show that the AD10 transgenic mice, housed in murine pathogen-free conditions (MPF-AD10 mice), also display a milder behavioral and neurodegenerative phenotype compared to the corresponding mice kept under conventional housing conditions (CV-AD10). As a first step toward the identification of mechanisms underlying this difference, a differential gene expression profiling was performed on brains from CV-AD10 and MPF-AD10 mice, showing a decrease of the immune response and neuroinflammation gene expression in MPF-AD10 mice. Results suggest that the activation of the immune response gene expression in the CV-AD10, in a microbially unprotected environment, might contribute to a more severe and progressive neurodegenerative phenotype, compared to the MPF mice.

Differences in serum cytokine levels between wild type mice and mice with a targeted mutation suggests necessity of using control littermates.

To enhance protection from pathogens, housing conditions have been improved constantly. We wanted to test whether various environmental conditions and caging systems affect serum cytokine levels of immunodeficient mice differently than they affect immunocompetent control animals. We compared serum cytokine levels of immunodeficient and immunocompetent mice kept in three different environments: a specific pathogen free (SPF) breeding barrier with open cages. An SPF experimental unit with individually ventilated cages. An experimental semi-barrier with open cages. Serum from Rag1(-/-), µMT(-/-), IFN-γR(-/-), IFN-γ(-/-), IL-4(-/-), the heterozygous controls and wild type C57BL/6 or BALB/c mice was analyzed for the presence of 10 cytokines (IL-1α, IL-2, IL-4, IL-5, IL-6, IL-10, IL-17, IFN-γ, TNF-α and GM-CSF). No major changes in cytokine levels were detected in mice exposed to different housing conditions. However, irrespective of immunodeficiency at 4 weeks of age a number of mice from the breeding colonies with a targeted mutation (TM), both -/- and +/- mice, showed a statistically significant elevation of some cytokines (primarily IL-1α, IL-5) when compared to wild type BALB/c and C57BL/6 mice. We conclude that under SPF conditions, immunodeficient mice can be kept either in open caging or IVC systems without affecting serum cytokine levels. The more important conclusion, however, stems from the observation that there is a significant difference in serum cytokine levels between wild type and mice carrying either one or two alleles of a targeted mutation (either -/- and +/- mice). This suggests an altered base-line inflammatory responsiveness in the TM-breeding colonies.

Early exposure to germs and the Hygiene Hypothesis.

A recent paper suggests that reduced exposure to germs results in the expansion of a cell type called natural killer T cells, which predisposes to colitis and asthma. Such a scenario could explain the Hygiene Hypothesis, which has been a puzzle for decades.

The continuum of intestinal CD4+ T cell adaptations in host-microbial mutualism.

How a mutualistic relationship between the intestinal microbiota and intestinal T cell compartments is established is important, as a breakdown of intestinal T cell homeostasis may cause inflammatory bowel diseases. A number of studies have shown that different bacterial species modulate the intestinal CD4(+) T cell compartment in different ways. We performed mechanistic in vivo studies that demonstrated the crucial requirement for regulatory T cells (Treg) and interleukin-10 (IL-10) in the induction of intestinal T cell homeostasis even following colonization with a completely benign microbiota. In the absence of a functional Treg response or IL-10 receptor signaling, the same bacteria that induced a Treg response in wild-type animals now induced T helper type 17 responses, without intestinal inflammation. Therefore, Treg, IL-10 and Th17 are crucial regulatory mechanisms in the intestine not only for controlling inflammation, but also to establish a continuum of CD4(+) T cell homeostasis upon commensal colonization.

Lymphocytes in neuroprotection, cognition and emotion: is intolerance really the answer?

Clinical, epidemiological and therapeutic studies indicate that some human depression is associated with proinflammatory cytokines, chronic inflammatory disorders, and inflammation-inducing lifestyle factors. Moreover depression can be induced by administration of proinflammatory cytokines, including IL-2 or IFN-α. However, recent studies in specific pathogen-free (SPF) rodents suggest a different--and potentially contradictory--relationship between immune processes and mental health. These studies suggest that effector T cells specific for central nervous system (CNS) antigens can assist recovery from an array of environmental insults ranging from nerve injury to psychological stress, while in contrast, regulatory T cells (Treg) oppose such recovery. Indeed, some reported effects of this so-called "protective autoimmunity" seem of direct relevance to depressive disorders. These findings pose a dilemma for those intending to manipulate inflammatory pathways as a treatment for depression. Should we administer anti-inflammatory treatments, or should we induce self-reactive T cells? We re-examine the rodent findings and outline immunological peculiarities of SPF rodents, the abnormal properties of their regulatory T cells, and the impact of gut microbiota. We find that "protective autoimmunity" is likely to be relevant only to very clean SPF animals that lack normal levels of activated T cells, CNS T cell traffic and mature Treg. The data indicate that even in SPF models the effectors of beneficial effects are not the proinflammatory autoimmune cells themselves, but rather unidentified regulatory cells. This reinterpretation of findings relevant to "protective autoimmunity" suggests that ongoing, and planned, clinical trials of anti-inflammatory strategies to treat depressive disorders are justified.