Effect of Human Infant Gut Microbiota on Mouse Behavior, Dendritic Complexity, and Myelination.

The mammalian gut microbiome influences numerous developmental processes. In human infants it has been linked with cognition, social skills, hormonal responses to stress, and brain connectivity. Yet, these associations are not necessarily causal. The present study tested whether two microbial stool communities, common in human infants, affected behavior, myelination, dendritic morphology, and spine density when used to colonize mouse models. Humanized animals were more like specific-pathogen free mice than germ-free mice for most phenotypes, although in males, both humanized groups were less social. Both humanized groups had thinner myelin sheaths in the hippocampus, than did germ-free animals. Humanized animals were similar to each other except for dendritic morphology and spine density where one group had greater dendritic length in the prefrontal cortex, greater dendritic volume in the nucleus accumbens, and greater spine density in both regions, compared to the other. Results add to a body of literature suggesting the gut microbiome impacts brain development. Teaser: Fecal transplants from human infants with highly abundant Bifidobacterium , an important inhabitant of the intestinal tract of breastfed newborns, may promote brain connectivity in mice.

Conjugative RP4 Plasmid-Mediated Transfer of Antibiotic Resistance Genes to Commensal and Multidrug-Resistant Enteric Bacteria In Vitro.

Many antibiotic-resistant bacteria carry resistance genes on conjugative plasmids that are transferable to commensals and pathogens. We determined the ability of multiple enteric bacteria to acquire and retransfer a broad-host-range plasmid RP4. We used human-derived commensal Escherichia coli LM715-1 carrying a chromosomal red fluorescent protein gene and green fluorescent protein (GFP)-labeled broad-host-range RP4 plasmid with ampR, tetR, and kanR in in vitro matings to rifampicin-resistant recipients, including Escherichia coli MG1655, Dec5α, Vibrio cholerae, Pseudomonas putida, Pseudomonas aeruginosa, Klebsiella pneumoniae, Citrobacter rodentium, and Salmonella Typhimurium. Transconjugants were quantified on selective media and confirmed using fluorescence microscopy and PCR for the GFP gene. The plasmid was transferred from E. coli LM715-1 to all tested recipients except P. aeruginosa. Transfer frequencies differed between specific donor-recipient pairings (10-2 to 10-8). Secondary retransfer of plasmid from transconjugants to E. coli LM715-1 occurred at frequencies from 10-2 to 10-7. A serial passage plasmid persistence assay showed plasmid loss over time in the absence of antibiotics, indicating that the plasmid imposed a fitness cost to its host, although some plasmid-bearing cells persisted for at least ten transfers. Thus, the RP4 plasmid can transfer to multiple clinically relevant bacterial species without antibiotic selection pressure.

Zoonotic Transmission of Campylobacter jejuni to Caretakers From Sick Pen Calves Carrying a Mixed Population of Strains With and Without Guillain Barré Syndrome-Associated Lipooligosaccharide Loci.

Campylobacter jejuni causes foodborne gastroenteritis and may trigger acute autoimmune sequelae including Guillain Barré Syndrome. Onset of neuromuscular paralysis is associated with exposure to C. jejuni lipooligosaccharide (LOS) classes A, B, C, D, and E that mimic and evoke antibodies against gangliosides on myelin and axons of peripheral nerves. Family members managing a Michigan dairy operation reported recurring C. jejuni gastroenteritis. Because dairy cattle are known to shed C. jejuni, we hypothesized that calves in the sick pen were the source of human infections. Fecal samples obtained from twenty-five calves, one dog, and one asymptomatic family member were cultured for Campylobacter. C. jejuni isolates were obtained from thirteen calves and the family member: C. coli from two calves, and C. hyointestinalis from two calves. Some calves had diarrhea; most were clinically normal. Typing of lipooligosaccharide biosynthetic loci showed that eight calf C. jejuni isolates fell into classes A, B, and C. Two calf isolates and the human isolate possessed LOS class E, associated mainly with enteric disease and rarely with Guillain Barré Syndrome. Multi-locus sequence typing, porA and flaA typing, and whole genome comparisons of the thirteen C. jejuni isolates indicated that the three LOS class E strains that included the human isolate were closely related, indicating zoonotic transmission. Whole-genome comparisons revealed that isolates differed in virulence gene content, particularly in loci encoding biosynthesis of surface structures. Family members experienced diarrheal illness repeatedly over 2 years, yet none experienced GBS despite exposure to calves carrying invasive C. jejuni with LOS known to elicit antiganglioside autoantibodies.

Campylobacter jejuni induces autoimmune peripheral neuropathy via Sialoadhesin and Interleukin-4 axes.

Campylobacter jejuni is a leading cause of gastroenteritis that has been causally linked with development of the autoimmune peripheral neuropathy Guillain Barré Syndrome (GBS). Previously, we showed that C. jejuni isolates from human enteritis patients induced Type1/17-cytokine dependent colitis in interleukin-10 (IL-10)-/- mice, while isolates from GBS patients colonized these mice without colitis but instead induced autoantibodies that cross-reacted with the sialylated oligosaccharide motifs on the LOS of GBS-associated C. jejuni and the peripheral nerve gangliosides. We show here that infection of IL-10-/- mice with the GBS but not the colitis isolate led to sciatic nerve inflammation and abnormal gait and hind limb movements, with character and timing consistent with this syndrome in humans. Autoantibody responses and associated nerve histologic changes were dependent on IL-4 production by CD4 T cells. We further show that Siglec-1 served as a central antigen presenting cell receptor mediating the uptake of the GBS isolates via interaction with the sialylated oligosaccharide motifs found specifically on the LOS of GBS-associated C. jejuni, and the ensuing T cell differentiation and autoantibody elicitation. Sialylated oligosaccharide motifs on the LOS of GBS-associated C. jejuni therefore acted as both the Siglec-1-ligand for phagocytosis, as well as the epitope for autoimmunity. Overall, we present a mouse model of an autoimmune disease induced directly by a bacterium that is dependent upon Siglec-1 and IL-4. We also demonstrate the negative regulatory role of IL-10 in C. jejuni induced autoimmunity and provide IL-4 and Siglec-1 blockade as potential therapeutic interventions against GBS.

Th1/Th17-mediated Immunity and Protection from Peripheral Neuropathy in Wildtype and IL10-/- BALB/c Mice Infected with a Guillain-Barré Syndrome-associated Campylobacter jejuni Strain.

Campylobacter jejuni is an important cause of bacterial gastroenteritis worldwide and is linked to Guillain-Barré syndrome (GBS), a debilitating postinfectious polyneuropathy. The immunopathogenesis of GBS involves the generation of antibodies that are cross reactive to C. jejuni lipooligosaccharide and structurally similar peripheral nerve gangliosides. Both the C. jejuni infecting strain and host factors contribute to GBS development. GBS pathogenesis is associated with Th2-mediated responses in patients. Moreover, induction of IgG1 antiganglioside antibodies in association with colonic Th2-mediated immune responses has been reported in C. jejuni-infected C57BL/6 IL10-/- mice at 4 to 6 wk after infection. We hypothesized that, due to their Th2 immunologic bias, BALB/c mice would develop autoantibodies and signs of peripheral neuropathy after infection with a GBS patient-derived strain of C. jejuni (strain 260.94). WT and IL10-/- BALB/c mice were orally inoculated with C. jejuni 260.94, phenotyped weekly for neurologic deficits, and euthanized after 5 wk. Immune responses were assessed as C. jejuni-specific and antiganglioside antibodies in plasma and cytokine production and histologic lesions in the proximal colon. Peripheral nerve lesions were assessed in dorsal root ganglia and their afferent nerve fibers by scoring immunohistochemically labeled macrophages through morphometry. C. jejuni 260.94 stably colonized both WT and IL10-/- mice and induced systemic Th1/Th17-mediated immune responses with significant increases in C. jejuni-specific IgG2a, IgG2b, and IgG3 plasma antibodies. However, C. jejuni 260.94 did not induce IgG1 antiganglioside antibodies, colitis, or neurologic deficits or peripheral nerve lesions in WT or IL10-/- mice. Both WT and IL10-/- BALB/c mice showed relative protection from development of Th2-mediated immunity and antiganglioside antibodies as compared with C57BL/6 IL10-/- mice. Therefore, BALB/c mice infected with C. jejuni 260.94 are not an effective disease model but provide the opportunity to study the role of immune mechanisms and host genetic background in the susceptibility to post infectious GBS.

Comparison of Effects of Trichuris muris and Spontaneous Colitis on the Proximal Colon Microbiota in C3H/HeJ and C3Bir IL10-/- Mice.

The nematode Trichuris muris has been shown to interact with specific enteric bacteria, but its effects on the composition of its host's microbial community are not fully understood. We hypothesized that Trichuris muris-infected mice would have altered colon microbiota as compared with uninfected mice. Colon histopathology and microbial community structure and composition were examined in mouse models of colitis (C3BirTLR4-/- IL10-/- and C3H/HeJ TLR4-/- IL10+/+ mice) with and without T. muris infection, in uninfected C3BirIL10-/- mice with and without spontaneous colitis, and in normal C3H/ HeJ mice. T. muris-infected mice developed colon lesions that were more severe than those seen in IL10-deficient mice. Ap- proximately 80% of infected IL10-/- mice had colon neutrophilic exudates, and some had extraintestinal worms and bacteria. The composition and structure of proximal colon microbiota were assessed by using terminal restriction fragment length polymorphism analysis targeting the bacterial 16S rRNA gene. Colon microbiota in C3BirIL10-/- and C3H/HeJ mice differed both qualitatively and quantitatively. Trichuris infection significantly altered the relative abundance of individual operational taxonomic units [OTU] but not the composition (presence or absence of OTU) of colon microbiota in the 2 mouse genotypes. When C3BirIL10-/- and C3H/HeJ mouse OTU were considered separately, Trichuris was found to affect the microbiota of C3BirIL10-/- mice but not of C3H/HeJ mice. Even though 34 of the 75 (45%) C3BirIL10-/- mice had spontaneous colitis, neither qualitative nor quantitative differences were detected in microbiota between colitic or noncolitic C3BirIL10-/- mice or noncolitic C3H/HeJ mice. Therefore, Trichuris-infected mice developed distinct microbial communities that were influenced by host background genes; these alterations cannot be attributed solely to colonic inflammation.

Experimental Evolution of Campylobacter jejuni Leads to Loss of Motility, rpoN (σ54) Deletion and Genome Reduction.

Evolution experiments in the laboratory have focused heavily on model organisms, often to the exclusion of clinically relevant pathogens. The foodborne bacterial pathogen Campylobacter jejuni belongs to a genus whose genomes are small compared to those of its closest genomic relative, the free-living genus Sulfurospirillum, suggesting genome reduction during the course of evolution to host association. In an in vitro experiment, C. jejuni serially passaged in rich medium in the laboratory exhibited loss of flagellar motility-an essential function for host colonization. At early time points the motility defect was often reversible, but after 35 days of serial culture, motility was irreversibly lost in most cells in 5 independently evolved populations. Population re-sequencing revealed disruptive mutations to genes in the flagellar transcriptional cascade, rpoN (σ54)-therefore disrupting the expression of the genes σ54 regulates-coupled with deletion of rpoN in all evolved lines. Additional mutations were detected in virulence-related loci. In separate in vivo experiments, we demonstrate that a phase variable (reversible) motility mutant carrying an adenine deletion within a homopolymeric tract resulting in truncation of the flagellar biosynthesis gene fliR was deficient for colonization in a C57BL/6 IL-10-/- mouse disease model. Re-insertion of an adenine residue partially restored motility and ability to colonize mice. Thus, a pathogenic C. jejuni strain was rapidly attenuated by experimental laboratory evolution and demonstrated genomic instability during this evolutionary process. The changes observed suggest C. jejuni is able to evolve in a novel environment through genome reduction as well as transition, transversion, and slip-strand mutations.

Suggested Curricular Guidelines for Musculoskeletal and Sports Medicine in Family Medicine Residency Training.

Patients often seek care from a family physician when they have a musculoskeletal injury or sport-related ailment. Family physicians must be adequately trained to provide this care. While general guidance is provided by the Accreditation Counsel for Graduate Medical Education (ACGME) it is left up to the individual programs to develop, implement, and execute their orthopedic and sports medicine curriculums. The American Academy of Family Physicians' (AAFP) Recommended Curriculum Guideline for Family Medicine Residents - Musculoskeletal and Sports Medicine provides a basic outline format for curriculum content and reference resources. The aim of this article is to elaborate on those training requirements and help programs to develop a curriculum implementation plan that will deliver a baseline level of competence for family medicine trainees.

An antibiotic depleted microbiome drives severe Campylobacter jejuni-mediated Type 1/17 colitis, Type 2 autoimmunity and neurologic sequelae in a mouse model.

The peripheral neuropathy Guillain-Barré Syndrome can follow Campylobacter jejuni infection when outer core lipooligosaccharides induce production of neurotoxic anti-ganglioside antibodies. We hypothesized that gut microbiota depletion with an antibiotic would increase C. jejuni colonization, severity of gastroenteritis, and GBS. Microbiota depletion increased C. jejuni colonization, invasion, and colitis with Type 1/17 T cells in gut lamina propria. It also stimulated Type 1/17 anti-C. jejuni and -antiganglioside-antibodies, Type 2 anti-C. jejuni and -antiganglioside antibodies, and neurologic phenotypes. Results indicate that both C. jejuni strain and gut microbiota affect development of inflammation and GBS and suggest that probiotics following C. jejuni infection may ameliorate inflammation and autoimmune disease.

A proteome-wide screen of Campylobacter jejuni using protein microarrays identifies novel and conformational antigens.

Campylobacter jejuni (C. jejuni) is a foodborne intestinal pathogen and major cause of gastroenteritis worldwide. C. jejuni proteins that are immunogenic have been sought for their potential use in the development of biomarkers, diagnostic assays, or subunit vaccines for humans or livestock. To identify new immunogenic C. jejuni proteins, we used a native protein microarray approach. A protein chip, with over 1400 individually purified GST-tagged C. jejuni proteins, representing over 86% of the proteome, was constructed to screen for antibody titers present in test sera raised against whole C. jejuni cells. Dual detection of GST signals was incorporated as a way of normalizing the variation of protein concentrations contributing to the antibody staining intensities. We detected strong signals to 102 C. jejuni antigens. In addition to antigens recognized by antiserum raised against C. jejuni, parallel experiments were conducted to identify antigens cross-reactive to antiserum raised against various serotypes of E. coli or Salmonella or to healthy human sera. This led to the identification of 34 antigens specifically recognized by the C. jejuni antiserum, only four of which were previously known. The chip approach also allowed identification of conformational antigens. We demonstrate in the case of Cj1621 that antigen signals are lost to denaturing conditions commonly used in other approaches to identify immunogens. Antigens identified in this study include those possessing sequence features indicative of cell surface localization, as well as those that do not. Together, our results indicate that the unbiased chip-based screen can help reveal the full repertoire of host antibodies against microbial proteomes.

Hip pain · difficulty walking · tenderness along the anteromedial thigh and groin · Dx?

A 14-year-old Caucasian boy presented to our clinic with a complaint of left anterior hip pain. The patient had been running during a flag football match when he suddenly developed a sharp, stabbing pain in his left hip. He said he felt a "pop" in his left groin while his left foot was planted and he was cutting to the right. The patient said this was followed by worsening pain with ambulation and hip flexion. The patient had considerable difficulty walking into the exam room. On physical examination, he had significant tenderness to palpation along the anteromedial thigh and groin. The patient's strength was 1/5 with left hip flexion. There was apparent muscle firing, but no significant leg movement. He had full passive range of motion and there was no soft-tissue swelling, erythema, or other integumentary changes.

Transplanted human fecal microbiota enhanced Guillain Barré syndrome autoantibody responses after Campylobacter jejuni infection in C57BL/6 mice.

BACKGROUND: Campylobacter jejuni is the leading antecedent infection to the autoimmune neuropathy Guillain-Barré syndrome (GBS), which is accompanied by an autoimmune anti-ganglioside antibody attack on peripheral nerves. Previously, we showed that contrasting immune responses mediate C. jejuni induced colitis and autoimmunity in interleukin-10 (IL-10)-deficient mice, dependent upon the infecting strain. Strains from colitis patients elicited T helper 1 (TH1)-dependent inflammatory responses while strains from GBS patients elicited TH2-dependent autoantibody production. Both syndromes were exacerbated by antibiotic depletion of the microbiota, but other factors controlling susceptibility to GBS are unknown. METHODS: Using 16S rRNA gene high-throughput sequencing, we examined whether structure of the gut microbial community alters host (1) gastrointestinal inflammation or (2) anti-ganglioside antibody responses after infection with C. jejuni strains from colitis or GBS patients. We compared these responses in C57BL/6 mice with either (1) stable human gut microbiota (Humicrobiota) transplants or (2) conventional mouse microbiota (Convmicrobiota). RESULTS: Inoculating germ-free C57BL/6 wild-type (WT) mice with a mixed human fecal slurry provided a murine model that stably passed its microbiota over >20 generations. Mice were housed in specific pathogen-free (SPF) facilities, while extra precautions of having caretakers wear sterile garb along with limited access ensured that no mouse pathogens were acquired. Humicrobiota conferred many changes upon the WT model in contrast to previous results, which showed only colonization with no disease after C. jejuni challenge. When compared to Convmicrobiota mice for susceptibility to C. jejuni enteric or GBS patient strains, infected Humicrobiota mice had (1) 10-100 fold increases in C. jejuni colonization of both strains, (2) pathologic change in draining lymph nodes but only mild changes in colon or cecal lamina propria, (3) significantly lower Th1/Th17-dependent anti-C. jejuni responses, (4) significantly higher IL-4 responses at 5 but not 7 weeks post infection (PI), (5) significantly higher Th2-dependent anti-C. jejuni responses, and (6) significantly elevated anti-ganglioside autoantibodies after C. jejuni infection. These responses in Humicrobiota mice were correlated with a dominant Bacteroidetes and Firmicutes microbiota. CONCLUSIONS: These data demonstrate that Humicrobiota altered host-pathogen interactions in infected mice, increasing colonization and Th-2 and autoimmune responses in a C. jejuni strain-dependent manner. Thus, microbiota composition is another factor controlling susceptibility to GBS.

Effects of antibiotic resistance (AR) and microbiota shifts on Campylobacter jejuni-mediated diseases.

Campylobacter jejuni is an important zoonotic pathogen recently designated a serious antimicrobial resistant (AR) threat. While most patients with C. jejuni experience hemorrhagic colitis, serious autoimmune conditions can follow including inflammatory bowel disease (IBD) and the acute neuropathy Guillain Barré Syndrome (GBS). This review examines inter-relationships among factors mediating C. jejuni diarrheal versus autoimmune disease especially AR C. jejuni and microbiome shifts. Because both susceptible and AR C. jejuni are acquired from animals or their products, we consider their role in harboring strains. Inter-relationships among factors mediating C. jejuni colonization, diarrheal and autoimmune disease include C. jejuni virulence factors and AR, the enteric microbiome, and host responses. Because AR C. jejuni have been suggested to affect the severity of disease, length of infections and propensity to develop GBS, it is important to understand how these interactions occur when strains are under selection by antimicrobials. More work is needed to elucidate host-pathogen interactions of AR C. jejuni compared with susceptible strains and how AR C. jejuni are maintained and evolve in animal reservoirs and the extent of transmission to humans. These knowledge gaps impair the development of effective strategies to prevent the emergence of AR C. jejuni in reservoir species and human populations.

Socioecological predictors of immune defences in wild spotted hyenas.

Social rank can profoundly affect many aspects of mammalian reproduction and stress physiology, but little is known about how immune function is affected by rank and other socio-ecological factors in free-living animals.In this study we examine the effects of sex, social rank, and reproductive status on immune function in long-lived carnivores that are routinely exposed to a plethora of pathogens, yet rarely show signs of disease.Here we show that two types of immune defenses, complement-mediated bacterial killing capacity (BKC) and total IgM, are positively correlated with social rank in wild hyenas, but that a third type, total IgG, does not vary with rank.Female spotted hyenas, which are socially dominant to males in this species, have higher BKC, and higher IgG and IgM concentrations, than do males.Immune defenses are lower in lactating than pregnant females, suggesting the immune defenses may be energetically costly.Serum cortisol and testosterone concentrations are not reliable predictors of basic immune defenses in wild female spotted hyenas.These results suggest that immune defenses are costly and multiple socioecological variables are important determinants of basic immune defenses among wild hyenas. Effects of these variables should be accounted for when attempting to understand disease ecology and immune function.

Antimicrobial Susceptibility Profiles of Human Campylobacter jejuni Isolates and Association with Phylogenetic Lineages.

Campylobacter jejuni is a zoonotic pathogen and the most common bacterial cause of human gastroenteritis worldwide. With the increase of antibiotic resistance to fluoroquinolones and macrolides, the drugs of choice for treatment, C. jejuni was recently classified as a serious antimicrobial resistant threat. Here, we characterized 94 C. jejuni isolates collected from patients at four Michigan hospitals in 2011 and 2012 to determine the frequency of resistance and association with phylogenetic lineages. The prevalence of resistance to fluoroquinolones (19.1%) and macrolides (2.1%) in this subset of C. jejuni isolates from Michigan was similar to national reports. High frequencies of fluoroquinolone-resistant C. jejuni isolates, however, were recovered from patients with a history of foreign travel. A high proportion of these resistant isolates were classified as multilocus sequence type (ST)-464, a fluoroquinolone-resistant lineage that recently emerged in Europe. A significantly higher prevalence of tetracycline-resistant C. jejuni was also found in Michigan and resistant isolates were more likely to represent ST-982, which has been previously recovered from ruminants and the environment in the U.S. Notably, patients with tetracycline-resistant C. jejuni infections were more likely to have contact with cattle. These outcomes prompt the need to monitor the dissemination and diversification of imported fluoroquinolone-resistant C. jejuni strains and to investigate the molecular epidemiology of C. jejuni recovered from cattle and farm environments to guide mitigation strategies.

Markedly Elevated Antibody Responses in Wild versus Captive Spotted Hyenas Show that Environmental and Ecological Factors Are Important Modulators of Immunity.

Evolutionary processes have shaped the vertebrate immune system over time, but proximal mechanisms control the onset, duration, and intensity of immune responses. Based on testing of the hygiene hypothesis, it is now well known that microbial exposure is important for proper development and regulation of the immune system. However, few studies have examined the differences between wild animals in their natural environments, in which they are typically exposed to a wide array of potential pathogens, and their conspecifics living in captivity. Wild spotted hyenas (Crocuta crocuta) are regularly exposed to myriad pathogens, but there is little evidence of disease-induced mortality in wild hyena populations, suggesting that immune defenses are robust in this species. Here we assessed differences in immune defenses between wild spotted hyenas that inhabit their natural savanna environment and captive hyenas that inhabit a captive environment where pathogen control programs are implemented. Importantly, the captive population of spotted hyenas was derived directly from the wild population and has been in captivity for less than four generations. Our results show that wild hyenas have significantly higher serum antibody concentrations, including total IgG and IgM, natural antibodies, and autoantibodies than do captive hyenas; there was no difference in the bacterial killing capacity of sera collected from captive and wild hyenas. The striking differences in serum antibody concentrations observed here suggest that complementing traditional immunology studies, with comparative studies of wild animals in their natural environment may help to uncover links between environment and immune function, and facilitate progress towards answering immunological questions associated with the hygiene hypothesis.

Metronidazole-but not IL-10 or prednisolone-rescues Trichuris muris infected C57BL/6 IL-10 deficient mice from severe disease.

Trichuris muris infected C57BL/6 mice are a frequently studied model of immune mediated resistance to helminths. Our objective was to characterize dose-dependent gastrointestinal (GI) disease and pathology due to Trichuris in C57BL/6 mice with varying degrees of IL-10 sufficiency. These mice can serve as a model for other animals (dogs, cattle) and humans where IL-10 polymorphisms have been associated with disease susceptibility and may affect susceptibility to whipworm. C57BL/6 IL-10(+/+), IL-10(+/-) and IL-10(-/-) mice were infected with T. muris (J strain) in a dose response study. T. muris produced dose-dependent disease in IL-10(-/-) mice. Ninety percent of mice receiving the high dose (75 ova) had severe disease necessitating early euthanasia, while the medium dose (50 ova) resulted in 100% early euthanasia of males/75% of females, and the low dose (25 ova) in 100% early euthanasia of males/25% of females. Having some IL-10 as in heterozygotes did not rescue all infected mice from effects of the high dose. 2/21 IL-10(-/-), 1/17 IL-10(+/-), and 0/17 IL-10(+/+) mice in the high dose group had severe peritonitis and extra-intestinal bacteria confirmed by fluorescent 16S rDNA analysis of peritoneal organ surfaces. Three of twenty one IL-10(-/-) had demonstrable extra-intestinal T. muris adults. Although free from viral pathogens, 12/21 IL-10(-/-), 6/17 IL-10(+/-), and 4/17 IL-10(+/+) infected mice had hepatitis, while control mice of all genotypes did not. Mice had evidence of inflammation of serosal surfaces of liver, spleen and GI tract even when extraintestinal Trichuris were not found. Blinded histopathology scoring revealed that even when infected IL-10(-/-) mice displayed few, if any, clinical signs, levels of gut inflammation did not vary significantly from those mice euthanized early due to severe disease. To examine whether antibiotics or corticosteroids could reverse severe disease and lesions, IL-10(-/-) mice infected with T. muris were treated with metronidazole or prednisolone prior to and throughout 40 days of infection. Mice given prednisolone had severe disease and lesions with the highest mortality rate. Mice given metronidazole had a significantly lower mortality rate than those given prednisolone, but GI lesions were of similar severity and distribution including peritonitis. Mortality was associated with extraintestinal worms and bacteria and further supported a role for enteric bacteria in this pathogenesis.

Characterization of Toll-like receptors 1-10 in spotted hyenas.

Previous research has shown that spotted hyenas (Crocuta crocuta) regularly survive exposure to deadly pathogens such as rabies, canine distemper virus, and anthrax, suggesting that they have robust immune defenses. Toll-like receptors (TLRs) recognize conserved molecular patterns and initiate a wide range of innate and adaptive immune responses. TLR genes are evolutionarily conserved, and assessing TLR expression in various tissues can provide insight into overall immunological organization and function. Studies of the hyena immune system have been minimal thus far due to the logistical and ethical challenges of sampling and preserving the immunological tissues of this and other long-lived, wild species. Tissue samples were opportunistically collected from captive hyenas humanely euthanized for a separate study. We developed primers to amplify partial sequences for TLRs 1-10, sequenced the amplicons, compared sequence identity to those in other mammals, and quantified TLR expression in lymph nodes, spleens, lungs, and pancreases. Results show that hyena TLR DNA and protein sequences are similar to TLRs in other mammals, and that TLRs 1-10 were expressed in all tissues tested. This information will be useful in the development of new assays to understand the interactions among the hyena immune system, pathogens, and the microbial communities that inhabit hyenas.