Renuka Subramaniam

Mycoplasma ovipneumoniae can predispose bighorn sheep to fatal Mannheimia haemolytica pneumonia.

Mycoplasma ovipneumoniae has been isolated from the lungs of pneumonic bighorn sheep (BHS). However experimental reproduction of fatal pneumonia in BHS with M. ovipneumoniae was not successful. Therefore the specific role, if any, of M. ovipneumoniae in BHS pneumonia is unclear. The objective of this study was to determine whether M. ovipneumoniae alone causes fatal pneumonia in BHS, or predisposes them to infection by Mannheimia haemolytica. We chose M. haemolytica for this study because of its isolation from pneumonic BHS, and its consistent ability to cause fatal pneumonia under experimental conditions. Since in vitro culture could attenuate virulence of M. ovipneumoniae, we used ceftiofur-treated lung homogenates from pneumonic BHS lambs or nasopharyngeal washings from M. ovipneumoniae-positive domestic sheep (DS) as the source of M. ovipneumoniae. Two adult BHS were inoculated intranasally with lung homogenates while two others received nasopharyngeal washings from DS. All BHS developed clinical signs of respiratory infection, but only one BHS died. The dead BHS had carried leukotoxin-positive M. haemolytica in the nasopharynx before the onset of this study. It is likely that M. ovipneumoniae colonization predisposed this BHS to fatal infection with the M. haemolytica already present in this animal. The remaining three BHS developed pneumonia and died 1-5 days following intranasal inoculation with M. haemolytica. On necropsy, lungs of all four BHS showed lesions characteristic of bronchopneumonia. M. haemolytica and M. ovipneumoniae were isolated from the lungs. These results suggest that M. ovipneumoniae alone may not cause fatal pneumonia in BHS, but can predispose them to fatal pneumonia due to M. haemolytica infection.

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 (APR). 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 ApR, 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.

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.

PCR ASSAY DETECTS MANNHEIMIA HAEMOLYTICA IN CULTURE-NEGATIVE PNEUMONIC LUNG TISSUES OF BIGHORN SHEEP (OVIS CANADENSIS) FROM OUTBREAKS IN THE WESTERN USA, 2009–2010

Abstract 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.

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.

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.

A Multivalent Mannheimia/Bibersteinia Vaccine Protects Bighorn Sheep Against Mannheimia haemolytica Challenge

ABSTRACT 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 × 105 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.

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.

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.