Experimental pneumonia induced by a Bordetella parapertussis-like organism in the ovine and murine lung : a thesis presented in partial fulfillment (70%) of the requirements for the degree of Master of Philosophy in Veterinary Pathology at Massey University

Thirty-four specific pathogen-free (SPF) Swiss mice were intranasally inoculated with a suspension containing about 3 x 10 7 colony-forming units (CFU)/ml of a B. parapertussis-like organism isolated from pneumonic ovine lung. Eleven per cent of the animals died between 2 and 3 days of inoculation and over 90% of infected mice developed a subacute bronchopneumonia morphologically similar to early lesions of naturally-occurring ovine chronic non-progressive pneumonia (CNP). The sequential pulmonary changes were examined by light microscopy and transmission electronmicroscopy from 12 hr to 29 days after inoculation. The early stages were characterized by alveolar septal congestion and oedema, focal intra-alveolar haemorrhage, and intra-alveolar and septal infiltration by neutrophils and macrophages. Later, hyperplasia of perivascular and peribronchiolar lymphoid tissue and the deposition of collagen in the interalveolar septa were prominent. The bronchial and bronchiolar epithelium remained intact throughout the experiment, but bronchiolar lumina became occluded by inflammatory exudate at an early stage. Ultrastructural changes consisted of the degeneration of the alveolar type I and type II epithelial cells and marked degeneration of alveolar macrophages. Pure cultures of the B. parapertussis-like organism were consistently recovered from mouse lungs 12 hr to 6 days after inoculation. Both intact and degenerating organisms were found free in alveolar spaces and within phagocytic vacuoles of alveolar macrophages. However, replication of organisms was not observed at any stage of infection and no special association was observed between organisms and the ciliated or non-ciliated respiratory epithelium. Injury to ovine respiratory tract was demonstrated when a similar bacterial suspension to that given to the mice was given by intratracheally to colostrum-deprived lambs. The lesions produced in the pulmonary parenchyma of the lambs were similar to those seen in both early naturally-occurring ovine CNP and the experimental infection with this organism in mice. They consisted of an acute mild tracheobronchitis, severe alveolar collapse and acute bronchopneumonia which developed within 24 hr and was most severe at 1 to 3 days after inoculation. Ultrastructurally, the alveolar epithelium exhibited extensive degenerative changes and necrosis of individual epithelial cells. Topographical studies revealed extensive coverage of the infected tracheobronchial epithelium with a dense layer of inflammatory cells mixed with mucus, and focal extrusion of ciliated cells. Occasionally, moderate numbers of the B. parapertussis-like coccobacilli were seen closely associated with cilia. Inoculated lambs showed a marked elevation in the numbers of cells in bronchoalveolar lavage 24 hr after infection. Up to 93% of the cells in the lavage at 24 hr were neutrophils. However, no close interation between phagocytic cells and the organises was detected. Many of the macrophages in the lavage exhibited cytoplasmic vacuolation from five days after inoculation onwards. Blood leucocyte and neutrophil counts in infected lambs gradually rose to reach peaks at five and three days after inoculation, respectively. The B. parapertussis-like organism was recovered in pure culture from the nasal cavity of lambs killed on days one, three, five and nine. The viable bacterial count in bronchoalveolar lavage fluid decreased from 24 hr to 5 days with almost complete elimination of organisms nine days after inoculation. The retention of the B. parapertussis-like organism in the mouse trachea was compared to that in the mouse lung from 0 to 48 hr after intranasal inoculation. Although there was greater bacterial deposition in the trachea than the lung there was a faster clearance from the trachea. At 48 hr after instillation, almost all organisms were eliminated from the trachea but about 45% of organisms were retained in the lung. The current investigation has shown that the B. parapertussis-like organism can infect SPF mice and colostrum-deprived lambs and induce a subacute bronchopneumonia. The morphological changes seen suggest that this organism has the potential to predispose the ovine respiratory tract to further infection by other microorganisms and that the organism itself may also be able to cause severe pulmonary damage. The relevance of these observations to the problem of CNP in sheep in the field has yet to be determined

Lymphotoxin-α Plays Only a Minor Role in Host Resistance to Respiratory Infection with Virulent Type A Francisella tularensis in Mice

This study examined the role of lymphotoxin (LT)-α in host defense against airborne infection with Francisella tularensis, a gram-negative facultative intracellular bacterium and the causative agent of tularemia. Following a low-dose aerosol infection with the highly virulent type A strain of F. tularensis, mice deficient in LTα (LTα−/−) consistently harbored approximately 10-fold fewer bacteria in their spleens at day 2 and 10-fold more bacteria in their lungs at day 4 than LTα+/+ mice. However, the mortality and median time to death were indistinguishable between the two mouse strains. In addition, the inflammatory responses to the infection, as reflected by the cytokine levels and leukocyte influx in the bronchoalveolar lavage fluid and histopathological analysis, were generally similar between LTα−/− and LTα+/+ mice. These data suggest that although LTα does not contribute significantly to the resistance and host responses of mice to airborne type A F. tularensis infection, it does play a subtle role in the multiplication/dissemination of F. tularensis

Role of Macrophages in Early Host Resistance to Respiratory Acinetobacter baumannii Infection

Acinetobacter baumannii is an emerging bacterial pathogen that causes nosocomial pneumonia and other infections. Although it is recognized as an increasing threat to immunocompromised patients, the mechanism of host defense against A. baumannii infection remains poorly understood. In this study, we examined the potential role of macrophages in host defense against A. baumannii infection using in vitro macrophage culture and the mouse model of intranasal (i.n.) infection. Large numbers of A. baumannii were taken up by alveolar macrophages in vivo as early as 4 h after i.n. inoculation. By 24 h, the infection induced significant recruitment and activation (enhanced expression of CD80, CD86 and MHC-II) of macrophages into bronchoalveolar spaces. In vitro cell culture studies showed that A. baumannii were phagocytosed by J774A.1 (J774) macrophage-like cells within 10 minutes of co-incubation, and this uptake was microfilament- and microtubule-dependent. Moreover, the viability of phagocytosed bacteria dropped significantly between 24 and 48 h after co-incubation. Infection of J774 cells by A. baumannii resulted in the production of large amounts of proinflammatory cytokines and chemokines, and moderate amounts of nitric oxide (NO). Prior treatment of J774 cells with NO inhibitors significantly suppressed their bactericidal efficacy (P<0.05). Most importantly, in vivo depletion of alveolar macrophages significantly enhanced the susceptibility of mice to i.n. A. baumannii challenge (P<0.01). These results indicate that macrophages may play an important role in early host defense against A. baumannii infection through the efficient phagocytosis and killing of A. baumannii to limit initial pathogen replication and the secretion of proinflammatory cytokines and chemokines for the rapid recruitment of other innate immune cells such as neutrophils

Characterization of HA and NA-containing VLPs produced in suspension cultures of HEK 293 cells

Virus like particles (VLPs) can be formulated into promising vaccines to prevent influenza infection. In addition of having a structure and composition that mimic the wild type virus, VLPs are safe since they are devoid of viral genes and consequently are not infectious. One approach to scale up the manufacturing of VLPs is to produce them in a serum-free suspension culture using a stable mammalian cell line. Importantly, with VLPs synthetized by mammalian cells, the post-translational modifications of the surface antigens should be similar to the wild type virus, and therefore should trigger a potent and specific immune response for the pathogen. As a proof of concept, we first established a cell line that was stably expressing hemagglutinin (HA) and neuraminidase (NA) proteins of influenza (subtype H1N1) using our patented cGMP human embryonic kidney (HEK293) cell line (clone 293SF-3F6). Transcription of the genes for these two glycoproteins was regulated by the inducible cumate transcription gene-switch. Next, to establish our capability to produce VLPs, we compared the formation of VLPs using these cells after forced expression of two scaffold proteins: Gag from the human immunodeficiency virus and M1 protein from influenza A (H1N1). In addition, monitoring of the VLPs was facilitated by fusing the Gag protein to the green fluorescent protein (GFP). VLP production was therefore initiated by transient transfection of plasmid encoding Gag or M1 and by addition of cumate to the culture medium. The VLPs secreted in the culture medium were recovered by ultracentrifugation on a sucrose cushion. The presence of HA an NA within the VLP fraction was demonstrated by western blot and quantified by dot blot. Interestingly, VLPs were produced more efficiently in the presence of Gag, indicating that Gag is a better scaffolding protein than M1 in this context. Under the electron microscope, the Gag-VLPs appeared as vesicles of 100 to 150 nm of diameter, containing a denser internal proteinous ring, which is a typical morphology for VLPs produced through Gag expression. The production of Gag-VLPs was also validated in a 3-L stirred tank bioreactor in serum-free medium. The immunogenicity of the VLPs is currently under investigation in a murine model for influenza. In conclusion, VLPs containing HA and NA can be manufactured in serum free suspension culture of HEK293 cells through forced expression of Gag. The efficacy of these VLPs for vaccination remains to be demonstrated

Climate-Smart Agriculture in Rwanda

The climate-smart agriculture (CSA) concept reflects an ambition to improve the integration of agriculture development and climate responsiveness. It aims to achieve food security and broader development goals under a changing climate and increasing food demand. CSA initiatives sustainably increase productivity, enhance resilience, and reduce/remove greenhouse gases (GHGs), and require planning to address tradeoffs and synergies between these three pillars: productivity, adaptation, and mitigation [1]. The priorities of different countries and stakeholders are reflected to achieve more efficient, effective, and equitable food systems that address challenges in environmental, social, and economic dimensions across productive landscapes. While the concept is new, and still evolving, many of the practices that make up CSA already exist worldwide and are used by farmers to cope with various production risks [2]. Mainstreaming CSA requires critical stocktaking of ongoing and promising practices for the future, and of institutional and financial enablers for CSA adoption. This country profile provides a snapshot of a developing baseline created to initiate discussion, both within countries and globally, about entry points for investing in CSA at scale

Fibrin-mediated Protection Against Infection-stimulated Immunopathology

Fibrin, a product of the blood coagulation cascade, accompanies many type 1 immune responses, including delayed-type hypersensitivity, autoimmunity, and graft rejection. In those settings, fibrin is thought to exacerbate inflammation and disease. Here, we evaluate roles for coagulation during infection with Toxoplasma gondii, a pathogen whose control requires robust type 1 immunity. We establish that fibrin prevents infection-stimulated blood loss, thereby performing a protective function that is essential for survival. Remarkably, fibrin does not simply protect against vascular damage caused directly by the infectious agent, but rather, protects against hemorrhage evoked by interferon-γ, a critical mediator of type 1 immunity. This finding, to our knowledge, is the first to document a beneficial role for coagulation during type 1 immunity, and suggests that fibrin deposition protects host tissue from collateral damage caused by the immune system as it combats infection

Anti-CD25 antibody-mediated depletion of effector T cell populations enhances susceptibility of mice to acute but not chronic Toxoplasma gondii infection.

Natural regulatory T cells (Tregs) constitutively express the IL-2R alpha-chain (CD25) on their surface. Consequently, administration of anti-CD25 Abs is a commonly used technique to deplete Treg populations in vivo. However, activated effector T cells may also transiently express CD25, and are thus also potential targets for anti-CD25 Abs. In this study using Toxoplasma gondii as a model proinflammatory infection, we have examined the capacity of anti-CD25 Abs to target effector T cell populations during an inflammatory episode, to determine to what extent that this action may modulate the outcome of disease. Anti-CD25 Ab-treated C57BL/6 mice displayed significantly reduced CD4(+) T cell IFN-gamma production during acute T. gondii infection and exhibited reduced weight loss and liver pathology during early acute infection; aspects of infection previously associated with effector CD4(+) T cell responses. In agreement, anti-CD25 Ab administration impaired parasite control and caused mice to succumb to infection during late acute/early chronic stages of infection with elevated tissue parasite burdens. In contrast, anti-CD25 Ab treatment of mice with established chronic infections did not markedly affect brain parasite burdens, suggesting that protective T cell populations do not express CD25 during chronic stages of T. gondii infection. In summary, we have demonstrated that anti-CD25 Abs may directly abrogate effector T cell responses during an inflammatory episode, highlighting important limitations of the use of anti-CD25 Ab administration to examine Treg function during inflammatory settings

Pan-HA antibodies for influenza detection and quantification

The influenza virus imposes a heavy burden for society in terms of health and economy. Influenza is an elusive enveloped virus due to antigenic shift and drift of two surface proteins: neuraminidase (NA) and hemagglutinin (HA). As a result, new strains emerge every year which require seasonal vaccination for protection. Furthermore, large vaccine quantities are urgently needed in case of pandemics. Theoretically, vaccines against a new strain can be manufactured in as little as three weeks with certain platforms and technologies. However, vaccine quantification and release are still relying on the use of the Single Radial Immunodiffusion (SRID) assay using a strain-specific antibody to calculate HA concentration. This is a major limitation because it can take up to three months to generate the reagents necessary to run the SRID assay, including the strain-specific antibody. Hence, one of the major hurdles in the process of influenza vaccine production is the quantification of HA which is critical to establish proper dosing. To circumvent the need for strain-specific antibodies, we have produced two monoclonal antibodies (F211-11H12-3 and F211-10A9-2) against a highly conserved peptide sequence found within the HA molecule (1). Multiple strains belonging to 13 different influenza A subtypes, as well as 6 strains belonging to B lineages were detected by Western blot and dot blot. Overall, mAb F211-11H12-3 recognizes preferentially influenza A subtype 1, while the mAb F211-10A9-2 has a higher affinity for influenza A subtype 2. Therefore, all strains tested could be detected when both mAb are combined and used as a cocktail. Next, we performed quantitative dot blots by generating a standard curve ranging from 160ng/ml to 20µg/ml HA. This method is simple, easy to implement and highly reproducible. In-process samples as well as purified material can be quantified by dot blot after denaturation with urea. Even though the SRID is the only assay approved by regulatory agencies, quantitative dot blots can be used during manufacturing to optimize and monitor the production process. Finally, ELISA is widely used for quantification and preliminary data demonstrates that samples can be quantified with the pan-HA mAbs. In conclusion, a pan-HA antibody cocktail was generated against a highly conserved peptide sequence of influenza. Viruses produced in eggs and mammalian cells from 40 different strains were detected by Western blot. Reproducible quantification was achieved by dot blot using the two mAbs and an appropriate calibrating standard. The combination of pan-HA antibodies with an immunoassay such as the dot blot assay could accelerate process development and help establish new generation quantification methods for influenza. As the field is looking for flexible and versatile solutions to shift away from the SRID assay and strain-specific antibodies, the development of broad-spectrum antibodies offers a long-awaited alternative. 1) Chun et al, Universal antibodies and their applications to the quantitative determination of virtually all subtypes of the influenza A viral hemagglutinins, Vaccine (26), pp 6068-6076, 2008

In vitro production and immunogenicity of a Clostridium difficile spore-specific BclA3 glycopeptide conjugate vaccine

The BclA3 glycoprotein is a major component of the exosporangial layer of Clostridium difficile spores and in this study we demonstrate that this glycoprotein is a major spore surface associated antigen. Here, we confirm the role of SgtA glycosyltransferase (SgtA GT) in BclA3 glycosylation and recapitulate this process by expressing and purifying SgtA GT fused to MalE, the maltose binding protein from Escherichia coli. In vitro assays using the recombinant enzyme and BclA3 synthetic peptides demonstrated that SgtA GT was responsible for the addition of β-O-linked GlcNAc to threonine residues of each synthetic peptide. These peptide sequences were selected from the central, collagen repeat region of the BclA3 protein. Following optimization of SgtA GT activity, we generated sufficient glycopeptide (10 mg) to allow conjugation to KLH (keyhole limpet hemocyanin) protein. Glycosylated and unglycosylated versions of these conjugates were then used as antigens to immunize rabbits and mice. Immune responses to each of the conjugates were examined by Enzyme Linked Immunosorbent Assay ELISA. Additionally, the BclA3 conjugated peptide and glycopeptide were used as antigens in an ELISA assay with serum raised against formalin-killed spores. Only the glycopeptide was recognized by anti-spore polyclonal immune serum demonstrating that the glycan moiety is a predominant spore-associated surface antigen. To determine whether antibodies to these peptides could modify persistence of spores within the gut, animals immunized intranasally with either the KLH-glycopeptide or KLH-peptide conjugate in the presence of cholera toxin, were challenged with R20291 spores. Although specific antibodies were raised to both antigens, immunization did not provide any protection against acute or recurrent disease