Weight and organ specific immune cell profiling of sleeve gastrectomy in mice.

OBJECTIVE: Sleeve gastrectomy (SG) has profound, immediate weight-loss independent effects on obesity related diabetes (T2D). Our prior studies have shown that immunologic remodeling may play a part in this metabolic improvement. However, to date, little is known about how the major immune cell populations change following SG and whether these are weight loss dependent. METHODS: Using mass cytometry with time of flight analysis (CyTOF), we broadly quantified the organ-specific immune cell repertoire induced by SG from splenic, jejunal, ileal, colonic, and hepatic lymphocyte fractions. Surgeries were performed in both diet-induced obese (DIO), insulin resistant mice and lean mice, which leads to sustained and non-sustained weight loss in SG animals compared to shams, respectively. Intergroup comparisons allow understanding of the relative contribution of diet, weight-loss, and surgery on immune profiling. Conserved immune changes represent surgery-specific, weight-independent, and diet-independent phenotypic changes. RESULTS: Initial analysis by way of visualization of t-distributed stochastic neighbor embedding analysis revealed changes in the B cell compartment following SG in both DIO and lean mice compared to Sham animals. In depth, traditional gating showed a shift within the splenic B cell compartment toward innate-like phenotype. There was a 1.3-fold reduction in follicular B cells within DIO SG (14% absolute reduction; p = 0.009) and lean SG (15% absolute reduction; p = 0.031) animals with a significant increase in innate-like B cell subsets in DIO SG mice(2.2 to 4.3-fold increase; p < 0.05). There was a similar trend toward increased innate B cell subsets in lean SG mice. There was a concomitant increase in multiple circulating immunoglobulin classes in both models. Further, lean (p = 0.009) and DIO SG animals (p = 0.015) had a conserved 5.5-fold and 5.7-fold increase, respectively, in splenic neutrophils and tendency toward M2 macrophage polarization. CONCLUSIONS: SG induces surgery-specific, weight-loss independent immune cells changes that have been previously linked to improved glucose metabolism. This immune phenotype may be a major contributor to post SG physiology. Characterizing the complex immune milieu following SG is an important step toward understanding the physiology of SG and the potential therapies therein.

A Review of Selected IBD Biomarkers: From Animal Models to Bedside.

Inflammatory bowel disease (IBD) is a dysregulated inflammatory condition induced by multiple factors. The etiology of IBD is largely unknown, and the disease progression and prognosis are variable and unpredictable with uncontrolled disease behavior. Monitoring the status of chronic colitis closely is challenging for physicians, because the assessment of disease activity and severity require invasive methods. Using laboratory biomarkers may provide a useful alternative to invasive methods in the diagnosis and management of IBD. Furthermore, patients with ulcerative colitis or Crohn's disease are also at risk of developing cancer. Annual colonoscopies can help lower the risk for developing colorectal cancer. However, laboratory biomarkers may also be helpful as non-invasive indicators in predicting treatment responses, improving prognosis, and predicting possible tumors. This review addresses selected laboratory biomarkers (including ANCA, chitinase 3-like 1, S100A12/RAGE, calprotectin, and TNF/TNFR2), which are identified by utilizing two well-accepted animal models of colitis, dextran sodium sulfate-induced and T cell receptor alpha knockout colitis models. In addition to being useful for monitoring disease severity, these biomarkers are associated with therapeutic strategies. The factors may regulate the initiation and perpetuation of inflammatory factors in the gut.

A microbial metabolite remodels the gut-liver axis following bariatric surgery.

Bariatric surgery is the most effective treatment for type 2 diabetes and is associated with changes in gut metabolites. Previous work uncovered a gut-restricted TGR5 agonist with anti-diabetic properties-cholic acid-7-sulfate (CA7S)-that is elevated following sleeve gastrectomy (SG). Here, we elucidate a microbiome-dependent pathway by which SG increases CA7S production. We show that a microbial metabolite, lithocholic acid (LCA), is increased in murine portal veins post-SG and by activating the vitamin D receptor, induces hepatic mSult2A1/hSULT2A expression to drive CA7S production. An SG-induced shift in the microbiome increases gut expression of the bile acid transporters Asbt and Ostα, which in turn facilitate selective transport of LCA across the gut epithelium. Cecal microbiota transplant from SG animals is sufficient to recreate the pathway in germ-free (GF) animals. Activation of this gut-liver pathway leads to CA7S synthesis and GLP-1 secretion, causally connecting a microbial metabolite with the improvement of diabetic phenotypes.

Bariatric surgery reveals a gut-restricted TGR5 agonist with anti-diabetic effects.

Bariatric surgery, the most effective treatment for obesity and type 2 diabetes, is associated with increased levels of the incretin hormone glucagon-like peptide-1 (GLP-1) and changes in levels of circulating bile acids. The levels of individual bile acids in the gastrointestinal (GI) tract after surgery have, however, remained largely unstudied. Using ultra-high performance liquid chromatography-mass spectrometry-based quantification, we observed an increase in an endogenous bile acid, cholic acid-7-sulfate (CA7S), in the GI tract of both mice and humans after sleeve gastrectomy. We show that CA7S is a Takeda G-protein receptor 5 (TGR5) agonist that increases Tgr5 expression and induces GLP-1 secretion. Furthermore, CA7S administration increases glucose tolerance in insulin-resistant mice in a TGR5-dependent manner. CA7S remains gut restricted, minimizing off-target effects previously observed for TGR5 agonists absorbed into the circulation. By studying changes in individual metabolites after surgery, the present study has revealed a naturally occurring TGR5 agonist that exerts systemic glucoregulatory effects while remaining confined to the gut.

Early changes in immune cell metabolism and function are a hallmark of sleeve gastrectomy: a prospective human study.

OBJECTIVE: To characterize longitudinal changes in blood biomarkers, leukocyte composition, and gene expression following laparoscopic sleeve gastrectomy (LSG). BACKGROUND: LSG is an effective treatment for obesity, leading to sustainable weight loss and improvements in obesity-related co-morbidities and inflammatory profiles. However, the effects of LSG on immune function and metabolism remain uncertain. METHODS: Prospective data was collected from 23 enrolled human subjects from a single institution. Parameters of weight, co-morbidities, and trends in blood biomarkers and leukocyte subsets were observed from pre-operative baseline to one year in three-month follow-up intervals. RNA-sequencing was performed on pairs of whole blood samples from the first six subjects of the study (baseline and three months post-surgery) to identify genome-wide gene expression changes associated with undergoing LSG. RESULTS: LSG led to a significant decrease in mean total body weight loss (18.1%) at three months and among diabetic subjects a reduction in HbA1c. Improvements in clinical inflammatory and hormonal biomarkers were demonstrated as early as three months after LSG. A reduction in neutrophil-lymphocyte ratio was observed, driven by a reduction in absolute neutrophil counts. Gene set enrichment analyses of differential whole blood gene expression demonstrated that after three months, LSG induced transcriptomic changes not only in inflammatory cytokine pathways but also in several key metabolic pathways related to energy metabolism. CONCLUSIONS: LSG induces significant changes in the composition and metabolism of immune cells as early as three months post-operatively. Further evaluation is required of bariatric surgery's effects on immunometabolism and consequences for host defense and metabolic disease.

Sleeve gastrectomy enhances glucose utilization and remodels adipose tissue independent of weight loss.

Sleeve gastrectomy (SG) induces weight loss-independent improvements in glucose homeostasis by unknown mechanisms. We sought to identify the metabolic adaptations responsible for these improvements. Nonobese C57BL/6J mice on standard chow underwent SG or sham surgery. Functional testing and indirect calorimetry were used to capture metabolic phenotypes. Tissue-specific glucose uptake was assessed by 18-fluorodeoxyglucose (18-FDG) PET/computed tomography, and RNA sequencing was used for gene-expression analysis. In this model, SG induced durable improvements in glucose tolerance in the absence of changes in weight, body composition, or food intake. Indirect calorimetry revealed that SG increased the average respiratory exchange ratio toward 1.0, indicating a weight-independent, systemic shift to carbohydrate utilization. Following SG, orally administered 18-FDG preferentially localized to white adipose depots, showing tissue-specific increases in glucose utilization induced by surgery. Transcriptional analysis with RNA sequencing demonstrated that increased glucose uptake in the visceral adipose tissue was associated with upregulation in transcriptional pathways involved in energy metabolism, adipocyte maturation, and adaptive and innate immune cell chemotaxis and differentiation. SG induces a rapid, weight loss-independent shift toward glucose utilization and transcriptional remodeling of metabolic and immune pathways in visceral adipose tissue. Continued study of this early post-SG physiology may lead to a better understanding of the anti-diabetic mechanisms of bariatric surgery.

Sleeve Gastrectomy and Roux-en-Y Gastric Bypass Attenuate Pro-inflammatory Small Intestinal Cytokine Signatures.

BACKGROUND: Bariatric surgery rapidly induces improvements in type 2 diabetes (T2D) in concert with reduction in systemic markers of inflammation. The impact of bariatric surgery on local intestinal immunity is not known. We hypothesize that sleeve gastrectomy (SG) and gastric bypass (RYGB) surgeries resolve obesity-induced intestinal inflammation, thereby promoting T2D resolution. METHODS: SG and RYGB, or control surgery was performed in SD rats (n = 4-6/group). Key cytokines involved in insulin resistance (TNF-α, IFN-γ), inflammasome activation (IL-1ß, IL-18), inflammation resolution (IL-10, IL-33), and Th17 cell responses (IL-17, IL-23) were measured by qPCR in mucosal scrapings of jejunum at 4 weeks post-surgery. Intestinal cytokine expressions were correlated with weight change, systemic and portal glucose, and insulin levels in response to an enteral glucose load. RESULTS: SG downregulated IL-17 and IL-23 in both proximal and distal jejunum, and IFN-γ was reduced only in distal jejunum (p < 0.05). Jejunal IL-17 and IL-23 expression correlated positively with weight changes after SG (0.93 and 0.98, respectively; p < 0.05). Changes in IFN-γ correlated strongly with insulin levels in portal and systemic circulation (0.99 and 0.95, respectively, p < 0.05). As with SG, IFN-γ, IL-17, and IL-23 were significantly reduced by RYGB. RYGB also reduced TNF-α and IL-18 and increased IL-33 levels (p < 0.05). CONCLUSIONS: RYGB and SG reduce expression of pro-inflammatory cytokines IL-17, IL-23, and IFN-γ in the jejunum. RYGB showed attenuation of additional pro-inflammatory cytokines and enhanced expression of IL-33. Post-surgical changes in intestinal IL-17, IL-23, and IFN-γ correlate strongly with changes in weight and glucose-triggered insulin responses.

Boosting efferocytosis in alveolar space using BCG vaccine to protect host against influenza pneumonia.

Efferocytosis by alveolar phagocytes (APs) is pivotal in maintenance of lung homeostasis. Increased efferocytosis by APs results in protection against lethal acute lung injury due to pulmonary infections whereas defective efferocytosis by APs results in chronic lung inflammation. In this report, we show that pulmonary delivery of Bacillus Calmette-Guerin (BCG) significantly enhances efferocytosis by APs. Increased efferocytosis by APs maintains lung homeostasis and protects mice against lethal influenza pneumonia. Intranasally treated wild type C57Bl/6 (WT) mice with BCG showed significant increase in APs efferocytosis in vivo compared to their PBS-treated counterparts. All BCG-treated WT mice survived lethal influenza A virus (IAV) infection whereas all PBS-treated mice succumbed. BCG-induced resistance was abrogated by depleting AP prior to IAV infection. BCG treatment increased uptake, and digestion/removal of apoptotic cells by APs. BCG significantly increased the expression of TIM4 on APs and increased expression of Rab5 and Rab7. We demonstrated that increased efferocytosis by APs through pulmonary delivery of BCG initiated rapid clearance of apoptotic cells from the alveolar space, maintained lung homeostasis, reduced inflammation and protected host against lethal IAV pneumonia.

Mechanistic roles of epithelial and immune cell signaling during the development of colitis-associated cancer.

To date, substantial evidence has shown a significant association between inflammatory bowel diseases (IBD) and development of colitis-associated cancer (CAC). The incidence/prevalence of IBD is higher in western countries including the US, Australia, and the UK. Although CAC development is generally characterized by stepwise accumulation of genetic as well as epigenetic changes, precise mechanisms of how chronic inflammation leads to the development of CAC are largely unknown. Preceding intestinal inflammation is one of the major influential factors for CAC tumorigenesis. Mucosal immune responses including activation of aberrant signaling pathways both in innate and adaptive immune cells play a pivotal role in CAC. Tumor progression and metastasis are shaped by a tightly controlled tumor microenvironment which is orchestrated by several immune cells and stromal cells including macrophages, neutrophils, dendritic cells, myeloid derived suppressor cells, T cells, and myofibroblasts. In this article, we will discuss the contributing factors of epithelial as well as immune cell signaling in initiation of CAC tumorigenesis and mucosal immune regulatory factors in the colonic tumor microenvironment. In depth understanding of these factors is necessary to develop novel anti-inflammatory and anti-cancer therapies for CAC in the near future.

Chitinase 3-like 1 induces survival and proliferation of intestinal epithelial cells during chronic inflammation and colitis-associated cancer by regulating S100A9.

Many host-factors are inducibly expressed during the development of inflammatory bowel disease (IBD), each having their unique properties, such as immune activation, bacterial clearance, and tissue repair/remodeling. Dysregulation/imbalance of these factors may have pathogenic effects that can contribute to colitis-associated cancer (CAC). Previous reports showed that IBD patients inducibly express colonic chitinase 3-like 1 (CHI3L1) that is further upregulated during CAC development. However, little is known about the direct pathogenic involvement of CHI3L1 in vivo. Here we demonstrate that CHI3L1 (aka Brp39) knockout (KO) mice treated with azoxymethane (AOM)/dextran sulphate sodium (DSS) developed severe colitis but lesser incidence of CAC as compared to that in wild-type (WT) mice. Highest CHI3L1 expression was found during the chronic phase of colitis, rather than the acute phase, and is essential to promote intestinal epithelial cell (IEC) proliferation in vivo. This CHI3L1-mediated cell proliferation/survival involves partial downregulation of the pro-apoptotic S100A9 protein that is highly expressed during the acute phase of colitis, by binding to the S100A9 receptor, RAGE (Receptor for Advanced Glycation End products). This interaction disrupts the S100A9-associated expression positive feedback loop during early immune activation, creating a CHI3L1hi S100A9low colonic environment, especially in the later phase of colitis, which promotes cell proliferation/survival of both normal IECs and tumor cells.

Plasminogen activator inhibitor-1 in cigarette smoke exposure and influenza A virus infection-induced lung injury.

Parenchymal lung inflammation and airway and alveolar epithelial cell apoptosis are associated with cigarette smoke exposure (CSE), which contributes to chronic obstructive pulmonary disease (COPD). Epidemiological studies indicate that people exposed to chronic cigarette smoke with or without COPD are more susceptible to influenza A virus (IAV) infection. We found increased p53, PAI-1 and apoptosis in AECs, with accumulation of macrophages and neutrophils in the lungs of patients with COPD. In Wild-type (WT) mice with passive CSE (PCSE), p53 and PAI-1 expression and apoptosis were increased in AECs as was lung inflammation, while those lacking p53 or PAI-1 resisted AEC apoptosis and lung inflammation. Further, inhibition of p53-mediated induction of PAI-1 by treatment of WT mice with caveolin-1 scaffolding domain peptide (CSP) reduced PCSE-induced lung inflammation and reversed PCSE-induced suppression of eosinophil-associated RNase1 (EAR1). Competitive inhibition of the p53-PAI-1 mRNA interaction by expressing p53-binding 3'UTR sequences of PAI-1 mRNA likewise suppressed CS-induced PAI-1 and AEC apoptosis and restored EAR1 expression. Consistent with PCSE-induced lung injury, IAV infection increased p53, PAI-1 and apoptosis in AECs in association with pulmonary inflammation. Lung inflammation induced by PCSE was worsened by subsequent exposure to IAV. Mice lacking PAI-1 that were exposed to IAV showed minimal viral burden based on M2 antigen and hemagglutination analyses, whereas transgenic mice that overexpress PAI-1 without PCSE showed increased M2 antigen and inflammation after IAV infection. These observations indicate that increased PAI-1 expression promotes AEC apoptosis and exacerbates lung inflammation induced by IAV following PCSE.

Delivery of GM-CSF to Protect against Influenza Pneumonia.

BACKGROUND: Since adaptive immunity is thought to be central to immunity against influenza A virus (IAV) pneumonias, preventive strategies have focused primarily on vaccines. However, vaccine efficacy has been variable, in part because of antigenic shift and drift in circulating influenza viruses. Recent studies have highlighted the importance of innate immunity in protecting against influenza. METHODS: Granulocyte-macrophage colony stimulating factor (GM-CSF) contributes to maturation of mononuclear phagocytes, enhancing their capacity for phagocytosis and cytokine production. RESULTS: Overexpression of granulocyte macrophage-colony stimulating factor (GM-CSF) in the lung of transgenic mice provides remarkable protection against IAV, which depends on alveolar macrophages (AM). In this study, we report that pulmonary delivery of GM-CSF to wild type young and aged mice abrogated mortality from IAV. CONCLUSION: We also demonstrate that protection is species specific and human GM-CSF do not protect the mice nor stimulates mouse immunity. We also show that IAV-induced lung injury is the culprit for side-effects of GM-CSF in treating mice after IAV infection, and introduce a novel strategy to deliver the GM-CSF to and retain it in the alveolar space even after IAV infection.

PCR assay detects Mannheimia haemolytica in culture-negative pneumonic lung tissues of bighorn sheep (Ovis canadensis) from outbreaks in the western USA, 2009-2010.

Mannheimia haemolytica consistently causes severe bronchopneumonia and rapid death of bighorn sheep (Ovis canadensis) under experimental conditions. However, Bibersteinia trehalosi and Pasteurella multocida have been isolated from pneumonic bighorn lung tissues more frequently than M. haemolytica by culture-based methods. We hypothesized that assays more sensitive than culture would detect M. haemolytica in pneumonic lung tissues more accurately. Therefore, our first objective was to develop a PCR assay specific for M. haemolytica and use it to determine if this organism was present in the pneumonic lungs of bighorns during the 2009-2010 outbreaks in Montana, Nevada, and Washington, USA. Mannheimia haemolytica was detected by the species-specific PCR assay in 77% of archived pneumonic lung tissues that were negative by culture. Leukotoxin-negative M. haemolytica does not cause fatal pneumonia in bighorns. Therefore, our second objective was to determine if the leukotoxin gene was also present in the lung tissues as a means of determining the leukotoxicity of M. haemolytica that were present in the lungs. The leukotoxin-specific PCR assay detected leukotoxin gene in 91% of lung tissues that were negative for M. haemolytica by culture. Mycoplasma ovipneumoniae, an organism associated with bighorn pneumonia, was detected in 65% of pneumonic bighorn lung tissues by PCR or culture. A PCR assessment of distribution of these pathogens in the nasopharynx of healthy bighorns from populations that did not experience an all-age die-off in the past 20 yr revealed that M. ovipneumoniae was present in 31% of the animals whereas leukotoxin-positive M. haemolytica was present in only 4%. Taken together, these results indicate that culture-based methods are not reliable for detection of M. haemolytica and that leukotoxin-positive M. haemolytica was a predominant etiologic agent of the pneumonia outbreaks of 2009-2010.

Protecting against post-influenza bacterial pneumonia by increasing phagocyte recruitment and ROS production.

Seasonal and especially pandemic influenza predispose patients to secondary bacterial pneumonias, which are a major cause of deaths and morbidity. Staphylococcus aureus is a particularly common and deadly form of post-influenza pneumonia, and increasing staphylococcal drug resistance makes the development of new therapies urgent. We explored an innate immune-mediated model of the lung to define novel mechanisms by which the host can be protected against secondary staphylococcal pneumonia after sub-lethal influenza infection. We found that stimulating the innate immunity in the lung by overexpression of GM-CSF will result in resistance to S. aureus pneumonia after sublethal influenza infection. Resistance was mediated by alveolar macrophages and neutrophils, and was associated with increased production of reactive oxygen species (ROS) by alveolar macrophages. Resistance was abrogated by treatment with agents that scavenged ROS. We conclude that stimulating innate immunity in the lung markedly reduces susceptibility to post-influenza staphylococcal pneumonia and that this may represent a novel immunomodulatory strategy for prevention and treatment of secondary bacterial pneumonia after influenza.

Role of Bibersteinia trehalosi, respiratory syncytial virus, and parainfluenza-3 virus in bighorn sheep pneumonia.

Pneumonic bighorn sheep (BHS) have been found to be culture- and/or sero-positive for Bibersteinia trehalosi, respiratory syncytial virus (RSV), and parainfluenza-3 virus (PI-3). The objective of this study was to determine whether these pathogens can cause fatal pneumonia in BHS. In the first study, two groups of four BHS each were intra-tracheally administered with leukotoxin-positive (Group I) or leukotoxin-negative (Group II) B. trehalosi. All four animals in Group I developed severe pneumonia, and two of them died within 3 days. The other two animals showed severe pneumonic lesions on euthanasia and necropsy. Animals in Group II neither died nor showed gross pneumonic lesions on necropsy, suggesting that leukotoxin-positive, but not leukotoxin-negative, B. trehalosi can cause fatal pneumonia in BHS. In the second study, two other groups of four BHS (Groups III and IV) were intra-nasally administered with a mixture of RSV and PI-3. Four days later, RSV/PI-3-inoculated Group IV and another group of four BHS (Group V, positive control) were intra-nasally administered with Mannheimia haemolytica, the pathogen that consistently causes fatal pneumonia in BHS. All four animals in group III developed pneumonia, but did not die during the study period. However all four animals in Group IV, and three animals in Group V developed severe pneumonia and died within two days of M. haemolytica inoculation. The fourth animal in Group V showed severe pneumonic lesions on euthanasia and necropsy. These findings suggest that RSV/PI-3 can cause non-fatal pneumonia, but are not necessary predisposing agents for M. haemolytica-caused pneumonia of BHS.

MHC class II DR allelic diversity in bighorn sheep.

We hypothesized that decreased diversity and/or unique polymorphisms in MHC class II alleles of bighorn sheep (BHS, Ovis canadensis) are responsible for lower titer of antibodies against Mannheimia haemolytica leukotoxin, in comparison to domestic sheep (DS, Ovis aries). To test this hypothesis, DRA and DRB transcripts from 24 captive BHS (Ovca-DRA and Ovca-DRB) were sequenced. Based on exon 2 (ß1 domain) sequences, eight different Ovca-DRB cDNA sequences were identified in BHS. Six of them were 100% identical to previously reported Ovca-DRB genomic DNA sequences. The new alleles DRB*23 and DRB*24, were closely related to two other Ovca-DRB exon 2 genomic DNA sequences. Nineteen out of 24 BHS (79%) Ovca-DRB exon 3 (ß2 domain) sequences were 100% identical to exon 3 sequence of DRB1 of DS (Ovar-DRB1). Ovca-DRA full length cDNA sequences exhibited >99% identity. Based upon exon 2 sequences, this BHS herd yielded higher Ovca-DRB allelic diversity than that reported in the previous study. Positively selected amino acid positions were identified in the peptide-binding groove of BHS and DS, but BHS showed more such sites. This highlights differing population histories, and may suggest differing needs for DR peptide-binding specificities. Presence of glutamine at position 52 (52Q) in some of the desert and captive BHS is predicted to alter the efficiency of DR dimerization, which may influence antigen presentation and T(h) cell activation. Functional assays with unique alleles should reveal whether the presentation of M. haemolytica leukotoxin peptides to T(h) cells by Ovca-DRB alleles is equivalent to that of Ovar-DRB1 alleles.

A multivalent Mannheimia-Bibersteinia vaccine protects bighorn sheep against Mannheimia haemolytica challenge.

Bighorn sheep (BHS) are more susceptible than domestic sheep (DS) to Mannheimia haemolytica pneumonia. Although both species carry M. haemolytica as a commensal bacterium in the nasopharynx, DS carry mostly leukotoxin (Lkt)-positive strains while BHS carry Lkt-negative strains. Consequently, antibodies to surface antigens and Lkt are present at much higher titers in DS than in BHS. The objective of this study was to determine whether repeated immunization of BHS with multivalent Mannheimia-Bibersteinia vaccine will protect them upon M. haemolytica challenge. Four BHS were vaccinated with a culture supernatant vaccine prepared from M. haemolytica serotypes A1 and A2 and Bibersteinia trehalosi serotype T10 on days 0, 21, 35, 49, and 77. Four other BHS were used as nonvaccinated controls. On the day of challenge, 12 days after the last immunization, the mean serum titers of Lkt-neutralizing antibodies and antibodies to surface antigens against M. haemolytica were 1:160 and 1:4,000, respectively. Following intranasal challenge with M. haemolytica A2 (1 × 10(5) CFU), all four control BHS died within 48 h. Necropsy revealed acute fibrinonecrotic pneumonia characteristic of M. haemolytica infection. None of the vaccinated BHS died during the 8 weeks postchallenge observation period. Radiography at 3 weeks postchallenge revealed no lung lesions in two vaccinated BHS and mild lesions in the other two, which resolved by 8 weeks postchallenge. These results indicate that if BHS can be induced to develop high titers of Lkt-neutralizing antibodies and antibodies to surface antigens, they are likely to survive M. haemolytica challenge which is likely to reduce the BHS population decline due to pneumonia.

Defective bacterial clearance is responsible for the enhanced lung pathology characteristic of Mannheimia haemolytica pneumonia in bighorn sheep.

The molecular and cellular basis for the enhanced lung pathology and mortality caused by Mannheimia haemolytica in bighorn sheep (BHS, Ovis canadenesis), in comparison to domestic sheep (DS, Ovis aries), is not clear. Polymorphonuclear leukocytes (PMNs) of BHS are four- to eight-fold more susceptible to M. haemolytica leukotoxin-induced cytolysis, which is likely to reduce the number of functional phagocytes in the lung. We hypothesized that enhanced lung pathology is due to defective clearance of M. haemolytica from the lungs of BHS. To test this hypothesis, M. haemolytica (1 × 10(7) colony forming units [cfu]) were inoculated intra-tracheally into three groups each of BHS and DS, which were euthanized and necropsied at 4, 12, and 18 h post-inoculation (hpi). Bacterial and leukocyte counts were performed on broncho-alveolar lavage fluid (BALF) collected at necropsy. BALF from BHS euthanized at 4 and 12 hpi contained a significantly higher number of M. haemolytica than that from DS. More importantly, DS did not have any bacteria in BALF at 18 hpi, while the BHS still had significant numbers. As expected, the BHS did exhibit more extensive lung lesions at 12 and 18 hpi when compared to DS. At 18 hpi, necrotic PMNs were observed in the lesional lung tissues of BHS, but not DS. Furthermore, BALF from BHS had significantly lower titers of antibodies to Lkt and surface antigens of M. haemolytica, than that of DS. These findings suggest that the enhanced pathology in BHS lungs is due to defective clearance of M. haemolytica from the lungs.

Transmission of Mannheimia haemolytica from domestic sheep (Ovis aries) to bighorn sheep (Ovis canadensis): unequivocal demonstration with green fluorescent protein-tagged organisms.

Previous studies demonstrated that bighorn sheep (Ovis canadensis) died of pneumonia when commingled with domestic sheep (Ovis aries) but did not conclusively prove that the responsible pathogens were transmitted from domestic to bighorn sheep. The objective of this study was to determine, unambiguously, whether Mannheimia haemolytica can be transmitted from domestic to bighorn sheep when they commingle. Four isolates of M. haemolytica were obtained from the pharynx of two of four domestic sheep and tagged with a plasmid carrying the genes for green fluorescent protein (GFP) and ampicillin resistance (AP(R)). Four domestic sheep, colonized with the tagged bacteria, were kept about 10 m apart from four bighorn sheep for 1 mo with no clinical signs of pneumonia observed in the bighorn sheep during that period. The domestic and bighorn sheep were then allowed to have fence-line contact for 2 mo. During that period, three bighorn sheep acquired the tagged bacteria from the domestic sheep. At the end of the 2 mo of fence-line contact, the animals were allowed to commingle. All four bighorn sheep died 2 days to 9 days following commingling. The lungs from all four bighorn sheep showed gross and histopathologic lesions characteristic of M. haemolytica pneumonia. Tagged M. haemolytica were isolated from all four bighorn sheep, as confirmed by growth in ampicillin-containing culture medium, PCR-amplification of genes encoding GFP and Ap(R), and immunofluorescent staining of GFP. These results unequivocally demonstrate transmission of M. haemolytica from domestic to bighorn sheep, resulting in pneumonia and death of bighorn sheep.