Patricia E. Shewen

Mannheimia haemolytica and bovine respiratory disease.

Abstract Mannheimia haemolytica is the principal bacterium isolated from respiratory disease in feedlot cattle and is a significant component of enzootic pneumonia in all neonatal calves. A commensal of the nasopharynx, M. haemolytica is an opportunist, gaining access to the lungs when host defenses are compromised by stress or infection with respiratory viruses or mycoplasma. Although several serotypes act as commensals, A1 and A6 are the most frequent isolates from pneumonic lungs. Potential virulence factors include adhesin, capsular polysaccharide, fimbriae, iron-regulated outer membrane proteins, leukotoxin (Lkt), lipopolysaccharide (LPS), lipoproteins, neuraminidase, sialoglycoprotease and transferrin-binding proteins. Of these, Lkt is pivotal in induction of pneumonia. Lkt-mediated infiltration and destruction of neutrophils and other leukocytes impairs bacterial clearance and contributes to development of fibrinous pneumonia. LPS may act synergistically with Lkt, enhancing its effects and contributing endotoxic activity. Antibiotics are employed extensively in the feedlot industry, both prophylactically and therapeutically, but their efficacy varies because of inconsistencies in diagnosis and treatment regimes and development of antibiotic resistance. Vaccines have been used for many decades, even though traditional bacterins failed to demonstrate protection and their use often enhanced disease in vaccinated animals. Modern vaccines use culture supernatants containing Lkt and other soluble antigens, or bacterial extracts, alone or combined with bacterins. These vaccines have 50–70% efficacy in prevention of M. haemolytica pneumonia. Effective control of M. haemolytica pneumonia is likely to require a combination of more definitive diagnosis, efficacious vaccines, therapeutic intervention and improved management practices.

Passive and active components of neonatal innate immune defenses.

Abstract Innate immune defenses are crucial for survival in the first days and weeks of life. At birth, newborns are confronted with a vast array of potentially pathogenic microorganisms that were not encountered in utero. At this age, cellular components of the adaptive immune system are in a naïve state and are slow to respond. Antibodies received from the dam are essential for defense, but represent a finite and dwindling resource. Innate components of the immune system detect pathogen-associated molecular patterns (PAMPs) on microorganisms (and their products) by means of pattern-recognition receptors (PRRs). Soluble mediators of the innate system such as complement proteins, pentraxins, collectins, ficolins, defensins, lactoferrin, lysozyme etc. can bind to structures on pathogens, leading to agglutination, interference with receptor binding, opsonization, neutralization, direct membrane damage and recruitment of additional soluble and cellular elements through inflammation. Cell-associated receptors such as the Toll-like receptors (TLRs) can activate cells and coordinate responses (both innate and adaptive). In this paper, accumulated knowledge of the receptors, soluble and cellular elements that contribute to innate defenses of young animals is reviewed. Research interest in this area has been intermittent, and the literature varies in quantity and quality. It is hoped that documentation of the limitations of our knowledge base will lead to more extensive and enlightening studies.

Towards Development of an Edible Vaccine against Bovine Pneumonic Pasteurellosis Using Transgenic White Clover Expressing a Mannheimia haemolytica A1 Leukotoxin 50 Fusion Protein

ABSTRACT Development of vaccines against bovine pneumonia pasteurellosis, or shipping fever, has focused mainly on Mannheimia haemolytica A1 leukotoxin (Lkt). In this study, the feasibility of expressing Lkt in a forage plant for use as an edible vaccine was investigated. Derivatives of the M. haemolytica Lkt in which the hydrophobic transmembrane domains were removed were made. Lkt66 retained its immunogenicity and was capable of eliciting an antibody response in rabbits that recognized and neutralized authentic Lkt. Genes encoding a shorter Lkt derivative, Lkt50, fused to a modified green fluorescent protein (mGFP5), were constructed for plant transformation. Constructs were screened by Western immunoblot analysis for their ability to express the fusion protein after agroinfiltration in tobacco. The fusion construct pBlkt50-mgfp5, which employs the cauliflower mosaic virus 35S promoter for transcription, was selected and introduced into white clover byAgrobacterium tumefaciens-mediated transformation. Transgenic lines of white clover were recovered, and expression of Lkt50-GFP was monitored and confirmed by laser confocal microscopy and Western immunoblot analysis. Lkt50-GFP was found to be stable in clover tissue after drying of the plant material at room temperature for 4 days. An extract containing Lkt50-GFP from white clover was able to induce an immune response in rabbits (via injection), and rabbit antisera recognized and neutralized authentic Lkt. This is the first demonstration of the expression of anM. haemolytica antigen in plants and paves the way for the development of transgenic plants expressing M. haemolytica antigens as an edible vaccine against bovine pneumonic pasteurellosis.

Oral Treatment of Chickens with Lactobacilli Influences Elicitation of Immune Responses

ABSTRACT Commensal microbes in the intestine are in constant interaction with host cells and play a role in shaping the immune system. Lactobacillus acidophilus, Lactobacillus reuteri, and Lactobacillus salivarius are members of the chicken intestinal microbiota and have been shown to induce different cytokine profiles in mononuclear cells in vitro. The objective of the present study was to examine the effects of these bacteria individually or in combination on the induction of antibody- and cell-mediated immune responses in vivo. The birds received lactobacilli weekly via oral gavage starting on day of hatch and subsequently, at 14 and 21 days, were immunized with sheep red blood cells (SRBC), keyhole limpet hemocyanin (KLH), Newcastle disease virus vaccine, and infectious bursal disease virus vaccine. Antibody responses in serum were measured weekly for 4 weeks beginning on the day of primary immunization. The cell-mediated immune response was evaluated at 21 days postimmunization by measurement of gamma interferon (IFN-γ) production in splenocytes stimulated with inactivated vaccine antigens. L. salivarius-treated birds had significantly more serum antibody to SRBC and KLH than birds that were not treated with probiotics. L. salivarius-treated birds also had decreased cell-mediated immune responses to recall antigen stimulation. L. reuteri treatment did not significantly affect the systemic immune response, while L. acidophilus treatment increased the antibody response to KLH. These results indicate that systemic antibody- and cell-mediated immune responses can be modulated by oral treatment with lactobacilli but that these bacteria may vary in their ability to modulate the immune response.

Evaluation of bacteriophages for prevention and treatment of diarrhea due to experimental enterotoxigenic Escherichia coli O149 infection of pigs.

The objective of this study was to determine the efficacy of selected phages individually and in combination in prevention and treatment of diarrhea due to experimental O149:H10:F4 enterotoxigenic Escherichia coli (ETEC) in weaned pigs. For prophylaxis, the phages were administered orally shortly after challenge, and for therapeutic use, were given 24h after challenge, following the onset of diarrhea. The parameters used to assess outcomes were weight change, duration of diarrhea, severity of diarrhea, composite diarrhea score, and extent of shedding of the challenge ETEC over 6 days. Six phages that were tested individually in a prophylactic mode were effective as determined by a significant change in each of the parameters, although the phages were not present at titres greater than 10(3)PFU/g of feces. A modified protocol involving pre-treatment of the pigs with florfenicol and oral administration of sodium bicarbonate prior to the ETEC challenge and phage administration resulted in high levels of phages in the feces. Using this protocol, a combination of three phages that was tested in the prophylactic mode significantly reduced the severity of diarrhea and the composite diarrhea score. A mixture of two phages given therapeutically significantly improved each of the outcome parameters, without perturbation of the total fecal E. coli flora. Enumeration of phages in feces after treatment indicated that the phages were replicating to high titres in the intestinal tract of ETEC infected pigs within 1-2 days before declining progressively. These findings indicate that the selected phages were effective in moderating the course of experimental O149:H10:F4 ETEC diarrhea in weaned pigs when given prophylactically or therapeutically.

Vaccination of neonates: problem and issues.

Many serious infectious diseases occur early in life; efficacious vaccination of neonates has been a longstanding goal in both human and veterinary medicine. Efforts to immunize in the first weeks of life, in various species, have had limited success in general. This has been attributed to a combination of immaturity of the neonatal immune system and interference by maternal antibodies. Most studies of neonatal immune responsiveness have been carried out in neonatal mice, or by examination of cellular components of human umbilical cord blood. Both approaches have their limitations. The current review describes factors, including corticosteroids, complement proteins, cytokines, maternal lymphocytes and antibodies, which may influence immune responses of neonates, comparing data from studies of domestic animals and humans. Neonates are highly dependent on passive (maternal) antibodies for protection against a wide range of pathogens. These maternal antibodies have been noted to interfere with active immune responses to many, but not all, vaccines. Various theories have been proposed to explain this phenomenon, including epitope masking, clearance of immune complexes and FcγRII mediated regulation of B cells. Remarkably, many studies examining the effects of passive antibodies on immune responses of adults, have demonstrated immune enhancing effects. The evidence for enhancing and suppressive effects of passive antibodies on antigen uptake, processing and regulation of lymphocyte responses is reviewed. Since maternal antibodies (as present in neonates) differ in subisotypes and affinity from the passive antibodies often used in experimental systems, here is a need for better experimental models investigating the effects of bona fide maternal antibodies on immune responses of neonates (not adult surrogates). Vaccines can be optimized for use in neonates - by making better use of existing vaccine technologies and by harnessing the potential of recent immunological and technological advances.

Plant-made vaccines: biotechnology and immunology in animal health.

Abstract The use of plants as production systems for vaccine antigens has been actively investigated over the last 15 years. The original research focused on the value of this expression system for oral delivery based on the hypothesis that plant-expressed antigens would be more stable within the digestive tract and would allow for the use of the oral route of administration to stimulate a mucosal immune response. However, while first conceived for utility via the oral route, plant-made antigens have also been studied as classical immunogens delivered via a needle to model animal systems. Antigens have been expressed in a number of whole plant and cell culture systems. Several alternative expression platforms have been developed to increase expression of antigens or to elicit preferred immunological responses. The biotechnological advances in plant expression and the immunological testing of these antigens will be reviewed in this paper focusing primarily on diseases of livestock and companion animals.

Clinical and serological evaluation of a Pasteurella haemolytica A1 capsular polysaccharide vaccine

The purified capsular polysaccharide (CPS) of Pasteurella haemolytica A1 was examined for its ability to protect cattle from experimental challenge with logarithmic-phase P. haemolytica. Several preparations of P. haemolytica antigens were utilized in the experiment including CPS, log-phase P. haemolytica culture supernatant, P. haemolytica recombinant leucotoxin (rLKt) and various combinations of the above. CPS alone or in combination with culture supernatant or rLkt elicited no protection; rather, administration of CPS was associated with a high incidence of anaphylaxis (36% of calves). Although a classical biphasic humoral immune response to CPS could be detected in all calves that received this compound, this T-dependent response was not correlated with resistance to experimental challenge. The complexity of protective immunity in pneumonic pasteurellosis is emphasized by this study, and clinical anaphylaxis associated with response to CPS may be implicated in the pathogenesis of disease.

Automated colorimetric assay for the detection of Pasteurella haemolytica leucotoxin

A rapid and easily performed colorimetric method has been adapted for the detection of the Pasteurella haemolytica leucotoxin using the lysis of bovine leucocyte BL-3 cells (a bovine lymphoid cell line obtained from Dr. G. Theilen, University of California, Davis). The method involves incubating BL-3 cells in the presence of P. haemolytica culture supernatant and assessing cell survival at the end of the assay by staining remaining BL-3 cells with the dye neutral red. Solubilization of the cells with concomitant dye release allows the assay to be read by measuring optical density (540 nm) with an automated spectrophotometer. This assay can also be used for the determination of neutralizing antiserum titres.

The effect of Pasteurella haemolytica A1 leukotoxic culture supernate on the in vitro proliferative response of bovine lymphocytes

The effect of sublethal concentrations of the Pasteurella haemolytica leukotoxic culture supernate on bovine lymphocyte blastogenesis was investigated. Blastogenesis in cultures stimulated with either concanavalin A (Con A) or pokeweed mitogen (PWM) was inhibited in the presence of the supernate, as was the response to purified protein derivative in lymphocytes from BCG-vaccinated cattle. Partially purified leukotoxin had a similar effect. Pre-incubation of the leukotoxic supernate with a polyclonal rabbit antiserum raised to the immunogenic molecule of recombinant leukotoxin (r LktA) abrogated this effect, implicating leukotoxin as the factor responsible for the inhibition. B cell enriched cultures tended to be more sensitive to leukotoxic effects than did T cell enriched cultures. Although only ruminant cells are susceptible to the lethal effects of P. haemolytica leukotoxin, the toxin did inhibit both Con A- and PWM-induced proliferation of human and dog lymphocytes. As well, at high leukotoxin doses, Con A-stimulated pig lymphocyte proliferation was reduced. Rabbit lymphocytes were not affected by leukotoxin in either Con A- or PWM-stimulated cultures.