A heterologous neuraminidase subtype strategy for the differentiation of infected and vaccinated animals (DIVA) for avian influenza virus using an alternative neuraminidase inhibition test.

The option of vaccinating poultry against avian influenza (AI) as a control tool is gaining greater acceptance by governments and the poultry industry worldwide. One disadvantage about vaccination with killed whole-virus vaccines is the resulting inability to use common serologic diagnostic tests for surveillance to identify infected flocks. There has been considerable effort to develop a reliable test for the differentiation of infected from vaccinated animals (DIVA). The heterologous neuraminidase (NA) subtype DIVA approach has been used with some success in the field accompanied by an ad hoc serologic test. The traditional NA inhibition (NI) test can be used for all nine NA subtypes, but it is time consuming, and it is not designed to screen large numbers of samples. In this study, a quantitative NI test using MUN (2'-[4-methylumbelliferyl]-alpha-D-Nacetylneuraminic acid sodium salt hydrate) as an NA substrate was investigated as an alternative to the traditional fetuin-based NI test in a heterologous neuraminidase DIVA strategy. Serum NI activity was determined in chickens administered different vaccines containing different H5 and NA subtypes and challenged with a highly pathogenic avian influenza (HPAI) H5N2 virus. Prior to challenge, the NI DIVA test clearly discriminated between chickens receiving vaccines containing different antigens (e.g., N8 or N9) from control birds that had no NA antibody. Some birds began to seroconvert 1 wk postchallenge, and 100% of the vaccinated birds had significant levels of N2 NI activity. This activity did not interfere with the presence of vaccine-induced NI activity against N8 or N9 subtypes. The level of N2-specific NI activity continued to increase to the last sampling date, 4 wk postchallenge, indicating the potential use for the heterologous NA-based DIVA strategy in the field.

Tumor necrosis factor-alpha enhances Haemophilus somnus lipooligosaccharide-induced apoptosis of bovine endothelial cells.

Haemophilus somnus lipooligosaccharide (LOS)-induced apoptosis of bovine pulmonary artery endothelial cells has been shown previously to be dependent on caspase-8 activation. Activation of caspase-8 can occur via a death receptor-dependent mechanism (e.g., TNF-alpha binding to TNF-alpha receptor 1 (TNF-R1)). In this study, we tested the hypothesis that TNF-alpha can enhance LOS-induced apoptosis of bovine endothelial cells. Addition of exogenous recombinant human TNF-alpha alone failed to cause apoptosis, or enhance LOS-induced apoptosis, of bovine endothelial cells. However, blocking de novo protein synthesis by addition of cycloheximide significantly enhanced apoptosis of bovine endothelial cells by TNF-alpha, LOS or TNF-alpha and LOS in combination. Conversely, addition of soluble recombinant human (sTNF-R1) diminished LOS-induced apoptosis. Overall, these data suggest that LOS-mediated apoptosis may be due, in part, to activation of a TNR-R1-dependent death pathway.

Stimulation of P2X receptors enhances lipooligosaccharide-mediated apoptosis of endothelial cells.

Exposure of endothelial cells to lipid A-containing molecules, such as lipopolysaccharide (LPS) or lipooligosaccharide (LOS), causes the release of purinergic compounds [e.g., adenosine 5'-triphosphate (ATP)] and can lead to apoptosis. The P2X family of purinergic receptors (e.g., P2X(7)) has been reported to modulate LPS signaling events and to participate in apoptosis. We investigated the role that P2X receptors play in the apoptosis that follows exposure of bovine endothelial cells to Haemophilus somnus LOS. Addition of P2X inhibitors, such as periodate-oxidized ATP (oATP) or pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium, significantly reduced LOS-induced apoptosis. Incubation of endothelial cells with apyrase, which degrades ATP, diminished LOS-induced apoptosis of endothelial cells. Concomitant addition of P2X agonists [e.g., 2',3'-(4-benzoyl)-benzoyl ATP or ATP] to LOS-treated endothelial cells significantly enhanced caspase-3 activation. The P2X antagonist oATP significantly blocked caspase-8 but not caspase-9 activation in LOS-treated endothelial cells. Together, these data indicate that stimulation of P2X receptors enhances LOS-induced apoptosis of endothelial cells, possibly as a result of endogenous release of ATP, which results in caspase-8 activation.

Enhanced 15-HPETE production during oxidant stress induces apoptosis of endothelial cells.

Oxidant stress plays an important role in the etiology of vascular diseases by increasing rates of endothelial cell apoptosis, but few data exist on the mechanisms involved. Using a unique model of oxidative stress based on selenium deficiency (-Se), the effects of altered eicosanoid production on bovine aortic endothelial cells (BAEC) apoptosis was evaluated. Oxidant stress significantly increased the immediate oxygenation product of arachidonic acid metabolized by the 15-lipoxygenase pathway, 15-hydroxyperoxyeicosatetraenoic acid (15-HPETE). Treatment of -Se BAEC with TNFalpha/cyclohexamide (CHX) exhibited elevated levels of apoptosis, which was significantly reduced by the addition of a specific 15-lipoxygenase inhibitor PD146176. Furthermore, the addition of 15-HPETE to PD146176-treated BAEC, partially restored TNF/CHX-induced apoptosis. Increased exposure to 15-HPETE induced apoptosis, as determined by internucleosomal DNA fragmentation, chromatin condensation, caspase-3 activation, and caspase-9 activation, which suggests mitochondrial dysfunction. The expression of Bcl-2 protein also was decreased in -Se BAEC. Addition of a caspase-9 inhibitor (LEHD-fmk) completely blocked 15-HPETE-induced chromatin condensation in -Se BAEC, suggesting that 15-HPETE-induced apoptosis is caspase-9 dependent. Increased apoptosis of BAEC as a result of oxidant stress and subsequent production of 15-HPETE may play a critical role in a variety of inflammatory based diseases.

Reactive oxygen and nitrogen intermediates contribute to Haemophilus somnus lipooligosaccharide-mediated apoptosis of bovine endothelial cells.

Although Haemophilus somnus causes septicemia and vasculitis in cattle, relatively little is known about how H. somnus affects endothelial cells in vitro. We previously reported that H. somnus lipooligosaccharide (LOS)-induced activation of caspases-3, -8 and -9, and apoptosis of bovine pulmonary artery endothelial cells (BPAEC) in vitro. Previous reports indicate that the generation of reactive oxygen species (ROS) or reactive nitrogen intermediates (RNI) can contribute to the induction of apoptosis. In the present study, we sought to determine whether ROS and RNI are involved in LOS-mediated apoptosis of BPAEC. We found that H. somnus LOS induced the generation of ROS in BPAEC, which was blocked by pretreatment with membrane permeable ROS scavengers, such as dimethylsulfoxide (DMSO) and allopurinol (AP). Addition of DMSO or AP significantly reduced H. somnus LOS-mediated caspase-3 activation. Addition of membrane impermeable ROS scavengers (e.g. catalase and superoxide dismutase), failed to block LOS-mediated caspase-3 activation, suggesting a role for intracellular generation of ROS in LOS-induced apoptosis of BPAEC. Addition of N(G)-nitro-L-arginine methyl ester (L-NAME) or aminoguanidine, which are selective inhibitors of nitric oxide synthase, blocked NO release and significantly reduced caspase-3 activation in LOS treated BPAEC. These data suggest H. somnus LOS triggers endogenous ROS and RNI production by endothelial cells, which contributes to apoptosis.

Complexities of the pathogenesis of Mannheimia haemolytica and Haemophilus somnus infections: challenges and potential opportunities for prevention?

Progress in producing improved vaccines against bacterial diseases of cattle is limited by an incomplete understanding of the pathogenesis of these agents. Our group has been involved in investigations of two members of the family Pasteurellaceae, Mannheimia haemolytica and Haemophilus somnus, which illustrate some of the complexities that must be confronted. Susceptibility to M. haemolytica is greatly increased during active viral respiratory infection, resulting in rapid onset of a severe and even lethal pleuropneumonia. Despite years of investigation, understanding of the mechanisms underlying this viral-bacterial synergism is incomplete. We have investigated the hypothesis that active viral infection increases the susceptibility of bovine leukocytes to the M. haemolytica leukotoxin by increasing the expression of or activating the beta2 integrin CD11a/CD18 (LFA-1) on the leukocyte surface. In vitro exposure to proinflammatory cytokines (i.e. interleukin-1beta, tumor necrosis factor-alpha and interferon-gamma) increases LFA-1 expression on bovine leukocytes, which in turn correlates with increased binding and responsiveness to the leukotoxin. Alveolar macrophages and peripheral blood leukocytes from cattle with active bovine herpesvirus-1 (BVH-1) infection are more susceptible to the lethal effects of the leukotoxin ex vivo than leukocytes from uninfected cattle. Likewise, in vitro incubation of bovine leukocytes with bovine herpesvirus 1 (BHV-1) potentiates LFA-1 expression and makes the cells more responsive to leukotoxin. A striking characteristic of H. somnus infection is its propensity to cause vasculitis. We have shown that H. somnus and its lipo-oligosaccharide (LOS) trigger caspase activation and apoptosis in bovine endothelial cells in vitro. This effect is associated with the production of reactive oxygen and nitrogen intermediates, and is amplified in the presence of platelets. The adverse effects of H. somnus LOS are mediated in part by activation of endothelial cell purinergic receptors such as P2X7. Further dissection of the pathways that lead to endothelial cell damage in response to H. somnus might help in the development of new preventive or therapeutic regimens. A more thorough understanding of M. haemolytica and H. somnus virulence factors and their interactions with the host might identify new targets for prevention of bovine respiratory disease.

Comparative efficacy of North American and antigenically matched reverse genetics derived H5N9 DIVA marker vaccines against highly pathogenic Asian H5N1 avian influenza viruses in chickens.

Highly pathogenic (HP) H5N1 avian influenza has become endemic in several countries in Asia and Africa, and vaccination is being widely used as a control tool. However, there is a need for efficacious vaccines preferably utilizing a DIVA (differentiate infected from vaccinated animals) marker strategy to allow for improved surveillance of influenza in vaccinated poultry. Using a reverse genetics approach, we generated Asian rgH5N9 vaccine strain deriving the hemagglutinin gene from A/chicken/Indonesia/7/2003 (H5N1) with modification of the cleavage site to be low pathogenic (LP) and N9 neuraminidase gene from the North American LP A/turkey/Wisconsin/1968 (H5N9) virus. The recombinant rgH5N9, A/turkey/Wisconsin/1968 (H5N9) A/chicken/Hidalgo/232/1994 (H5N2), and wild type HP A/chicken/Indonesia/7/2003 (H5N1) viruses were used to prepare inactivated oil-emulsified whole virus vaccines. Two weeks after vaccination, chickens were challenged with either Asian HP H5N1 viruses, A/chicken/Indonesia/7/2003 (W.H.O. clade 2.1) or A/chicken/Supranburi Thailand/2/2004 (W.H.O. clade 1.0). The H5 HA1 of the North American vaccine strains exhibited 12% amino acid differences including amino acid changes in the major antigenic sites as compared to the Asian HP H5N1 challenge viruses, serologically exhibited substantial antigenic difference, but still provided 100% protection from mortality. However, challenge virus shedding was significantly higher in chickens immunized with antigenically distinct American lineage vaccines as compared to the antigenically matched Asian rgH5N9 and the wild type Asian H5N1 vaccine. The antibody response to the heterologous subtype neuraminidase proteins were discriminated in vaccinated and infected chickens using a rapid fluorescent 2'-(4-methylumbelliferyl)-alpha-d-N-acetylneuraminic acid sodium salt as substrate for neuraminidase inhibition assay. This study demonstrates the value of using a vaccine containing antigenically matched H5 hemagglutinin for control of HP H5N1 avian influenza in poultry and the potential utility of a heterologous neuraminidase as a DIVA marker.

Protective avian influenza in ovo vaccination with non-replicating human adenovirus vector.

Protective immunity against avian influenza virus was elicited in chickens by single-dose in ovo vaccination with a non-replicating human adenovirus vector encoding an H5N9 avian influenza virus hemagglutinin. Vaccinated chickens were protected against both H5N1 (89% hemagglutinin homology; 68% protection) and H5N2 (94% hemagglutinin homology; 100% protection) highly pathogenic avian influenza virus challenges. This vaccine can be mass-administered using available robotic in ovo injectors which provide a major advantage over current vaccination regimens. In addition, this class of adenovirus-vectored vaccines can be produced rapidly with improved safety since they do not contain any replication-competent adenoviruses. Furthermore, this mode of vaccination is compatible with epidemiological surveys of natural avian influenza virus infections.

Signaling through interleukin-1 type 1 receptor diminishes Haemophilus somnus lipooligosaccharide-mediated apoptosis of endothelial cells.

During sepsis, endothelial cells are both a source and target of pro-inflammatory cytokines (e.g. IL-1alpha, IL-1beta, TNFalpha and others), which may be detrimental to vascular homeostasis. Our laboratory has demonstrated that Haemophilus somnus, a gram-negative pathogen of cattle that causes sepsis and vasculitis, and its lipooligosaccharide (LOS) induce caspases-3, -8 and -9 activation, and apoptosis of endothelial cells in vitro. In this study, we provide evidence that H. somnus LOS increases IL-1alpha and IL-1beta mRNA expression, and caspase-1 activation in endothelial cells. Addition of a caspase-1 inhibitor (YVAD), or incubation in a high extracellular potassium buffer (150 mM), reduced caspase-1 activation and significantly enhanced H. somnus LOS-mediated caspase-3 activation. Likewise, blocking the IL-1 type 1 receptor by addition of IL-receptor antagonist (IL-1ra) significantly enhanced LOS-mediated caspase-3 activation. Conversely, addition of exogenous recombinant bovine IL-1beta (100 ng/mL) to endothelial cells diminished LOS-mediated apoptosis. IL-1beta has been reported previously to protect numerous cell types from apoptosis by activating PI3 kinase/p-Akt signaling pathways. Addition of selective PI3 kinase inhibitors (e.g. wortmannin and LY294002) significantly enhanced LOS-mediated caspase-3 activation. Exposure of endothelial cells to IL-1beta or LOS increased pAkt protein as assessed by western blot. Overall, these results suggest that signaling through the IL-1 type 1 receptor diminishes H. somnus LOS-mediated apoptosis.

Bovine platelets activated by Haemophilus somnus and its LOS induce apoptosis in bovine endothelial cells.

Haemophilus somnus is a bacterial pathogen that causes respiratory disease and vasculitis in cattle. Thrombotic meningoencephalitis (TME) and other severe forms of H. somnus-mediated vascular disease are characterized histopathologically by vasculitis, thrombosis, and infiltration of polymorphonuclear cells. It has been reported previously that activated human platelets express CD40L, FasL and P-selectin (CD62P). We hypothesized that if these surface markers are up-regulated on bovine platelets after in vitro exposure to H. somnus and its lipooligosaccharide (LOS), they might contribute to endothelial cell damage. Using flow cytometry, we demonstrated low baseline expression of these molecules by bovine platelets and increased expression following in vitro stimulation with ADP, H. somnus or H. somnus LOS. H. somnus stimulated platelets were capable of causing apoptosis in endothelial cells as measured by Hoechst-33342 staining and caspase-3 activity. If these events occur in vivo, they might promote vascular damage and endothelial cell apoptosis, leading to the development of vasculitis and thrombosis that characterize bovine H. somnus infection.

Caspase activation during Haemophilus somnus lipooligosaccharide-mediated apoptosis of bovine endothelial cells.

Vasculitis is commonly seen during systemic Haemophilus somnus infections. Although, the mechanism of vascular damage is not completely understood, in a previous report we demonstrated that H. somnus and its lipooligosaccharide (LOS) induced apoptosis in bovine pulmonary artery endothelial cells in vitro. In the present study, we investigated the role of caspase activation in LOS-mediated apoptosis of bovine endothelial cells. Exposure to H. somnus LOS induced caspase-3 activation and chromatin condensation in endothelial cells. These responses were blocked by the addition of a pan-caspase inhibitor (z-VAD-fmk) or capase-3 inhibitor (DEVD-fmk). Incubation of endothelial cells with H. somnus LOS also induced activation of the initiator caspases, caspases-8 and 9, with the activity of the former increasing more rapidly than the latter. Addition of a caspase-8 inhibitor (IETD-fmk) significantly reduced LOS-mediated apoptosis, whereas, addition of a caspase-9 inhibitor (LEHD-fmk) had little effect. These data suggest that LOS-mediated activation of caspase-3 and apoptosis of endothelial cells is caspase-8 dependent.

Immunogenicity of chi4127 phoP- Salmonella enterica serovar Typhimurium in dogs.

Salmonellae are commonly isolated from dogs. The number of dogs infected with Salmonella spp. is surprisingly high and greater than the incidence of clinical disease would suggest. Salmonellosis is common in greyhound kennels. Morbidity can approach 100% in puppies and the mortality ranges to nearly 40%. To date, there has been little effort to evaluate the feasibility of a vaccine for control of this disease in dogs. In the studies described here, an attenuated strain of Salmonella enterica serovar Typhimurium (Se Typhimurium), chi4127, was capable of establishing a limited infection in dogs. The chi4127-attenuated salmonellae efficiently stimulated protective immune responses in serotype homologous, direct, oral challenge experiments. Morbidity in the wild-type-challenged dogs was 8.3% in immunized dogs but 100% in the non-vaccinated controls. In (9/12) control dogs, the disease involved both gastrointestinal and respiratory tracts with high fever (>40.2 degrees C) that persisted through 5 days after challenge. Serum IgG response against S. typhimurium lipopolysaccharide (LPS) significantly increased (P<0.01) in vaccinated dogs and in non-vaccinated dogs after challenge. The non-vaccinated dogs had 3 to 4 logs higher numbers of Se Typhimurium in splenic and hepatic tissue than did the vaccinated dogs. This particular attenuated strain has potential for use as a vaccine for canine salmonellosis.