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Mannheimia (Pasteurella) haemolytica A1 is the primary bacterial agent of bovine pneumonic pasteurellosis (shipping fever), which is characterized by acute lobar fibronecrotizing pneumonia with extensive peripheral blood neutrophil (PMN) infiltration in small airways and alveoli (4,39,47). Several virulence factors of M. haemolytica play an important role in the pathogenesis of pasteurellosis (7, 13). Foremost among these is a leukotoxin (LKT), whose effects are specific for ruminant leukocytes and platelets (2, 6, 9, 44). The M. haemolytica LKT is member of the repeats-in-toxin (RTX) family of gram-negative bacterial pore-forming exotoxins (46). Members of the RTX family have similar mechanisms of toxin production, secretion, and target cell intoxication (8, 45). Previously, it has been reported that other members of the RTX family bind to ␤ 2 -integrins on target cells (23). More recently, it has been demonstrated that M. haemolytica LKT binds to lymphocyte function-associated antigen 1 (LFA-1), a ␤ 2 -integrin (CD 11a/CD18) on bovine leukocytes (1,17,25,27). LKT binding to bovine leukocytes induces formation of pore-like structures in the plasma membrane, resulting in both activation of leukocytes and death by necrosis and apoptosis (14,18,24,29,34,40,43,45,53).For reasons that are not well understood, active viral infections can greatly enhance the susceptibility of cattle to M. haemolytica pneumonia (11,28,42,48,49). One mechanism that might be involved is the release of inflammatory cytokines during viral infection (33, 34). Inflammatory cytokines secreted by respiratory tract cells, such as interleukin 1 (IL-1␤), tumor necrosis factor alpha (TNF-␣), and gamma interferon (IFN-␥), can stimulate leukocyte migration and functional activation of ␤ 2 -integrins on lung leukocytes (10,35,38). Once M. haemolytica infection is established in the lung, the continued release of these inflammatory cytokines could be sustained by M. haemolytica virulence factors (i.e., LKT and lipopolysaccharide [LPS]) (15,21,22,30,50,51,52).PMNs are thought to contribute to the lung pathology observed in pneumonic pasteurellosis (4). PMN depletion reduces the severity of lung damage in experimentally infected cattle (4, 39). We hypothesized that inflammatory cytokines released during viral infection might increase surface expression or conformational activation of LFA-1 on bovine PMNs, thus amplifying their interaction with M. haemolytica LKT. In this study, we demonstrated increased expression of LFA-1 on bovine PMNs, as detected by flow cytometry, following incubation of PMNs with IL-1␤, TNF-␣, or IFN-␥. This in turn was reflected in increased LKT binding to, and cytotoxicity for, bovine PMNs. These observations suggest that the ability of inflammatory cytokines to increase surface expression or conformational activation of LFA-1 on bovine PMNs increases their interaction with M. haemolytica LKT. The outcome of the response might increase the severity of bovine pasteurellosis. MATERIALS AND METHODSPMN preparation. Peripheral blood ...

The present work evaluated the use of probiotics during the nursery rearing of the pink shrimp Farfantepenaeus brasiliensis, in a zero exchange aerobic heterotrophic culture system during 30 days. Three replicate tanks were randomly assigned to the following treatments: (1) Bacillus spp. mixture (Sanolife Pro‐‐W®), (2) Bacillus sp., Enterococcus sp., Lactobacillus spp. mixture (Biomin Start‐grow®), (3) Bacillus cereus var. toyoi and (4) control treatment (without probiotic addition). Bacteriological analysis monitored the abundance of presumptive Vibrio spp. in the water of experimental tanks. For the immunological analysis, shrimp haemolymph was collected to determine the granular haemocyte count and total protein concentration. Results showed that mean final weight and specific growth rate of shrimp in the probiotic treatments were significantly higher. Furthermore, shrimp reared in the probiotic treatments showed higher levels of total protein and granular haemocyte. The bacteriological analysis showed that the concentration of Vibrio spp. measured in probiotic treatment tanks was lower than that recorded in the control tanks.

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.

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The cattle tick, Rhipicephalus microplus, is regarded as the most economically important ectoparasite of livestock globally. Control is achieved primarily through the use of acaricides. This approach is hampered by the development of resistance to commercial acaricides among cattle tick populations. Vaccination against R. microplus infestation is another technology that can be integrated for effective cattle tick control. Proteins belonging to the Kunitz-BPTI family are abundant in cattle tick salivary glands, midgut, and ovaries. These organs are attractive targets for the development of a novel cattle tick vaccine. Efficacy assessment against cattle tick infestation in bovines using a vaccine containing the recombinant form of a member of the Kunitz family from R. microplus produced in a yeast expression system is reported for the first time here. The yeast Pichia pastoris was bioengineered to produce the recombinant version of a trypsin inhibitor that is expressed in cattle tick larvae (rRmLTI). Immunization with rRmLTI afforded 32% efficacy against R. microplus. The estimated molecular weight of rRmLTI was 46 kDa. Structural homology to the native form of the larval trypsin inhibitor was documented by recognition of rRmLTI in Western-blots using polyclonal antibodies from mice immunized with cattle tick larval extract or rRmLTI. Bioinformatics analysis of the partial nucleotide and deduced amino acid sequences indicated that the rRmLTI closely resembles BmTI-6, which is a three-headed Kunitz protein present in cattle tick ovary and fat tissue.

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