Shipping Fever Pneumonia Research Articles

Channel Formation by LktA of Mannheimia (Pasteurella) haemolytica in Lipid Bilayer Membranes and Comparison of Channel Properties with Other RTX-Cytolysins.

Cytolysin LktA is one of the major pathogenicity factors of Mannheimia haemolytica (formerly Pasteurella haemolytica) that is the cause of pasteurellosis, also known as shipping fever pneumonia, causing substantial loss of sheep and cattle during transport. LktA belongs to the family of RTX-toxins (Repeats in ToXins) that are produced as pathogenicity factors by a variety of Gram-negative bacteria. Sublytic concentrations of LktA cause inflammatory responses of ovine leukocytes. Higher concentrations result in formation of transmembrane channels in target cells that may cause cell lysis and apoptosis. In this study we investigated channel formation by LktA in artificial lipid bilayer membranes made of different lipids. LktA purified from culture supernatants by polyethylene glycol 4000 precipitation and lyophilization had to be activated to frequently form channels by solution in 6 M urea. The LktA channels had a single-channel conductance of about 60 pS in 0.1 M KCl, which is about one tenth of the conductance of most RTX-toxins with the exception of adenylate cyclase toxin of Bordetella pertussis. The LktA channels are highly cation-selective caused by negative net charges. The theoretical treatment of the conductance of LktA as a function of the bulk aqueous concentration allowed a rough estimate of the channel diameter, which is around 1.5 nm. The size of the LktA channel is discussed with respect to channels formed by other RTX-toxins. We present here the first investigation of LktA in a reconstituted system.

Bovine-like coronaviruses in domestic and wild ruminants.

Coronaviruses (CoVs) produce a wide spectrum of disease syndromes in different mammalian and avian host species. These viruses are well-recognized for their ability to change tissue tropism, to hurdle the interspecies barriers and to adapt ecological variations. It is predicted that the inherent genetic diversity of CoVs caused by accumulation of point mutations and high frequency of homologous recombination is the principal determinant of these competences. Several CoVs (e.g. Severe acute respiratory syndrome-CoV, Middle East respiratory syndrome-CoV) have been recorded to cross the interspecies barrier, inducing different disease conditions in variable animal hosts. Bovine CoV (BCoV) is a primary cause of gastroenteritis and respiratory disease in cattle calves, winter dysentery in lactating cows and shipping fever pneumonia in feedlot cattle. Although it has long been known as a restrictive cattle pathogen, CoVs that are closely related to BCoV have been recognized in dogs, humans and in other ruminant species. Biologic, antigenic and genetic analyses of the so-called 'bovine-like CoVs' proposed classification of these viruses as host-range variants rather than distinct virus species. In this review, the different bovine-like CoVs that have been identified in domesticated ruminants (water buffalo, sheep, goat, dromedary camel, llama and alpaca) and wild ruminants (deer, wild cattle, antelopes, giraffes and wild goats) are discussed in terms of epidemiology, transmission and virus characteristics. The presented data denote the importance of these viruses in the persistence of BCoV in nature, spread to new geographical zones, and continuous emergence of disease epidemics in cattle farms.

The hydrophilic proteins of lung surfactant as a prognostic marker in experimental pneumonia

Abstract BACKGROUND: SP-A and SP-D are hydrophilic proteins which regulate the inflammatory response of the lung. Pasteurella multocida is one of the most common bacteria isolated from calves suffering from shipping fever pneumonia, one of the most problems in dairy herds. OBJECTIVE: Evaluation of surfactant content may provide a valuable diagnostic tool for detection of calf pneumonia due to Pasteurella multocida and also state of treatment. METHODS: Ten Holstein-Frisian bull calves aged 4 months with body weight of 120 ± 5 kg were selected for study in two groups. The Pasteurella multocida (PMC66 Razi) was used in the present study for inducing pneumonia. The Bronchoalveolar lavage (BAL) process was done in selected calves. BAL fluid was collected and centrifuged and finally the sediment (crude surfactant) was reserved at -20˚C.The cytological evaluation and surfactant content was assayed by ELISA, TPL kit assay and HPLC. RESULTS: The serum levels of SP-A and SP-D in pneumonic group were significantly elevated. Although the increased Bronchoalveolar lavage fluid (BALF) level of SP-A in pneumonic cases was found as compared with the control animals, but the statistical analysis didn't show any significant differences between two groups. The level of SP-D in BALF of pneumonic group significantly elevated. The amount of Dipalmitoylphosphatidylcholine (DPPC) in pneumonic group decreased significantly in comparison control group. CONCLUSION: Pasteurella inducing pulmonary can changed the major component of lung surfactant which evaluation of these markers can be helpful as an appropriate tool in diagnostic state of pneumonia and healing.

Natural competence in Histophilus somni strain 2336

Histophilus somni is an etiologic agent of shipping fever pneumonia, myocarditis, and other systemic diseases of bovines. Virulence factors that have been identified in H. somni include biofilm formation, lipooligosaccharide phase variation, immunoglobulin binding proteins, survival in phagocytic cells, and many others. However, to identify the genes responsible for virulence, an efficient mutagenesis system is needed. Mutagenesis of H. somni using allelic exchange is difficult, likely due to its tight restriction modification system. Mutagenesis by natural transformation in Haemophilus influenzae is well established and shows a strong bias for fragments containing specific uptake signal sequences (USS) within the genome. We hypothesized that natural transformation may also be possible in H. somni strain 2336 because its genome is over-represented with H. influenzae USS (5′-AAGTGCGGT-3′) and contains most of the genes necessary for competence. H. somni strain 2336 was successfully transformed and mutated with genomic linear DNA from an H. somni mutant (738Δlob2a), which contains a kanamycin-resistance (KanR) gene and the USS within lob2A. Although most of the competence genes found in H. influenzae were present in H. somni, comD and the 5′ portion of comE were absent, which may account for the low transformation efficiency. The transformation efficiency of strain 2336 was greatest during mid-log growth phase and when cyclic adenosine monophosphate was added to the transformation medium. However, mutants were not isolated when strain 2336 was transformed with genomic DNA containing the same KanR gene from H. somni luxS or uspE mutants, which lack the USS in these specific genes. Shuttle vector pNS3K was also naturally transformed into strain 2336, though at a lower efficiency. However, natural transformation with either H. somni linear DNA (2336Δlob2A) or pNS3K was unsuccessful with H. somni commensal strain 129Pt and several other disease isolates.

Mannheimia haemolytica chimeric protein vaccine composed of the major surface-exposed epitope of outer membrane lipoprotein PlpE and the neutralizing epitope of leukotoxin

Mannheimia haemolytica is commonly identified in cattle with shipping fever pneumonia. Vaccines currently available do not provide complete protection against the disease. In an effort to develop a vaccine that delivers the immunogenic regions of leukotoxin (LKT) A and the outer membrane protein (OMP) PlpE, a total of four chimeric proteins were constructed. Mice were subcutaneously immunized with 25, 50 and 75 μg quantities of each chimeric protein. The specificity of the immune response was confirmed by Western blot analysis and enzyme-linked immunosorbent assays (ELISA). Moreover, the hyperimmune sera were bactericidal to M. haemolytica in the presence of complement and neutralized LKT. While all of the chimeric proteins induced some level of immune response two, SAC87 and SAC89, were most promising. These results demonstrate that a functional immune response against M. haemolytica can be induced by vaccination with recombinant chimeric proteins created from specific immunogenic regions of the LKT and PlpE proteins.

Impairment of innate immune responses of airway epithelium by infection with bovine viral diarrhea virus

Bovine viral diarrhea virus (BVDV) infection is an important risk factor for development of shipping fever pneumonia in feedlot cattle, and infects but does not cause morphologic evidence of damage to airway epithelial cells. We hypothesized that BVDV predisposes to bacterial pneumonia by impairing innate immune responses in airway epithelial cells. Primary cultures of bovine tracheal epithelial cells were infected with BVDV for 48 h, then stimulated with LPS for 16 h. Expression of tracheal antimicrobial peptide (TAP) and lingual antimicrobial peptide (LAP) mRNA was measured by quantitative RT-PCR, and lactoferrin concentrations were measured in culture supernatant by ELISA. BVDV infection had no detectable effect on the constitutive expression of TAP and LAP mRNA or lactoferrin concentration in culture supernatant. LPS treatment provoked a significant increase in TAP mRNA expression and lactoferrin concentration in the culture supernatant ( p < 0.01), and these effects were significantly ( p < 0.02, p < 0.01) abrogated by prior infection of the tracheal epithelial cells with the type 2 ncp-BVDV isolate. In contrast, infection with the type 1 ncp-BVDV isolate had no effect on TAP mRNA expression or lactoferrin secretion. LPS treatment induced a significant ( p < 0.001) upregulation of LAP mRNA expression, which was not significantly affected by prior infection with BVDV. These data indicate that infection with a type 2 BVDV isolate inhibits the LPS-induced upregulation of TAP mRNA expression and lactoferrin secretion by tracheal epithelial cells, suggesting a novel mechanism by which this virus abrogates respiratory innate immune responses and predisposes to bacterial pneumonia in cattle.

Effect of pH, temperature and nutrient limitations on growth and leukotoxin production by Mannheimia haemolytica in batch and continuous culture

Quantification of the effects of pH, temperature and nutrient limitations on the growth and leukotoxin (LKT) production parameters of Mannheimia haemolytica in batch and chemostat culture. Mannheimia haemolytica strains OVI-1 and PH12296 were grown aerobically in two semi-defined media. In amino acid-limited cultures, the LKT concentration and yield in terms of biomass (Y(LKT/x)) were up to eightfold greater than in carbon-limited cultures. Supplementing amino acid-limited chemostat cultures with cysteine, glutamine, ferric iron and manganese further enhanced the Y(LKT/x) values up to threefold. Supplementation of an amino acid-limited batch culture of M. haemolytica strain OVI-1 with these nutrients resulted in an LKT concentration of 1.77 g l(-1) that was 45-fold greater than that obtained in RPMI 1640 medium. Aerobiosis enhanced LKT production. High acetic acid concentrations were produced under carbon-sufficient conditions. The highest maximum specific growth rates were recorded in the range of pH 6.8 to 7.8 and 37 to 40 degrees C. An amino acid-limited culture medium greatly improved LKT production in aerobic batch culture, which could be further enhanced by supplementation with cysteine, glutamine, ferric iron and manganese. It was demonstrated that LKT production by M. haemolytica could be dramatically increased through manipulation of the culture medium composition, which could benefit the production of LKT-based vaccines against bovine shipping fever pneumonia.

Sequence diversity of the immunogenic outer membrane lipoprotein PlpE from Mannheimia haemolytica serotypes 1, 2, and 6

Mannheimia haemolytica serotype 1 (S1), S6 and S2 are the most common bacterial isolates found in shipping fever pneumonia in beef cattle. Currently used vaccines against M. haemolytica do not provide complete protection against the disease. Research with M. haemolytica outer membrane proteins (OMPs) has shown that antibodies to one particular OMP from S1, PlpE, may be important in immunity. Recombinant PlpE (rPlpE) is highly immunogenic in cattle, and the acquired immunity markedly enhanced resistance to experimental challenge. We previously demonstrated that the immunodominant epitope (R2) is located between residues 26 and 76 on the N-terminus of PlpE from a reference S1 strain ( Ayalew et al., 2004). This region consists of eight hexapeptide repeats. The potential of this epitope as a vaccine or supplement to commercial vaccines is dependant on its state of conservation amongst isolates of the three serotypes. To determine this, we sequenced plpE genes from 32 isolates. The sequences from S1 and S6 were identical with one exception. Substantial variation was observed among sequences from S2 strains, particularly in the R2 region of the protein. These variations in S2 isolates range from 3 to 28 hexapeptide repeats. Calculated molecular weight of PlpE from S1 and S6 isolates was 37 kDa, where as PlpE from S2 strains ranged from 30 to 50 kDa. These similarities and differences were demonstrated by western blot. Competitive binding assay was used to determine that antibody against rPlpE from S1 binds native PlpE on surfaces of both S1 and S2 cells.

Characterization of Immunodominant and Potentially Protective Epitopes of Mannheimia haemolytica Serotype 1 Outer Membrane Lipoprotein PlpE

Mannheimia haemolytica serotype 1 (S1) is the most common bacterial isolate found in shipping fever pneumonia in beef cattle. Currently used vaccines against M. haemolytica do not provide complete protection against the disease. Research with M. haemolytica outer membrane proteins (OMPs) has shown that antibodies to one particular OMP from S1, PlpE, may be important in immunity. In a recently published work, members of our laboratory showed that recombinant PlpE (rPlpE) is highly immunogenic when injected subcutaneously into cattle and that the acquired immunity markedly enhanced resistance to experimental challenge (A. W. Confer, S. Ayalew, R. J. Panciera, M. Montelongo, L. C. Whitworth, and J. D. Hammer, Vaccine 21:2821-2829, 2003). The objective of this work was to identify epitopes of PlpE that are responsible for inducing the immune response. Western blot analysis of a series of rPlpE with nested deletions on both termini with bovine anti-PlpE hyperimmune sera showed that the immunodominant region is located close to the N terminus of PlpE. Fine epitope mapping, in which an array of overlapping 13-mer synthetic peptides attached to a derivatized cellulose membrane was probed with various affinity-purified anti-PlpE antibodies, identified eight highly reactive regions, of which region 2 (R2) was identified as the specific epitope. The R2 region is comprised of eight imperfect repeats of a hexapeptide (QAQNAP) and is located between residues 26 and 76. Complement-mediated bactericidal activity of affinity-purified anti-PlpE bovine antibodies confirmed that antibodies directed against the R2 region are effective in killing M. haemolytica.

Pneumonia in horses induced by intrapulmonary inoculation of Streptococcus equi subsp. zooepidemicus.

To evaluate the possibility that Streptococcus equi subsp. zooepidemicus (S.z) the causative bacterial agent of equine shipping fever pneumonia (ESFP), as well as to investigate its pathogenesis, 10 horses (seven Thoroughbreds and three Anglo-Arab species, ranging from 2-4 years in age) were experimentally inoculated, via an endoscope, into bronchus of the lung lobe with a dose of 30 ml of 1-7 x 10(8) CFU/ml of S.z. After inoculation, autopsy and pathological examinations were sequentially conducted 30 min, 1, 2, 3, 4, 17, 20 hr and 2 weeks later. Pneumonia induced by the intrapulmonary inoculation of S.z was characterized by small purulent pneumonic foci in the inoculated areas. With the lapse of time, these foci developed into serous hemorrhagic pneumonia, hemorrhagic purulent pneumonia, and then purulent, coagulation necrotic pneumonia. These pathomorphological characteristics of experimental pneumonia closely resemble those naturally occurring ESFP. There is strong evidence that S.z. is implicated as a causal factor in ESFP. S.z. grew in the mucus, exudate, and pulmonary effusions. Further, the bacteria showed resistance against phagocytosis by pulmonary alveolar macrophages (PAM) and neutrophils. Inhibition of PAM and neutrophil function is considered to be important in the development of pneumonia. With the progression of the disease, the neutrophils often adhered to the endothelial surface of the alveolar capillary lumen and played a role in generating coagulation necrosis of lung tissues.

Use of operon fusions in Mannheimia haemolytica to identify environmental and cis-acting regulators of leukotoxin transcription.

The leukotoxin of Mannheimia haemolytica is an important virulence factor that contributes to much of the pathology observed in the lungs of animals with bovine shipping fever pneumonia. We believe that identification of factors that regulate leukotoxin expression may provide insight into M. haemolytica pathogenicity. The DNA sequence upstream of the leukotoxin operon is divergently shared by P(lapT), which transcribes an arginine permease gene. The intergenic region contains several elements that are potential sites for transcriptional modulation of the promoters. We have developed plasmid-borne chloramphenicol acetyltransferase (cat) operon fusions, as well as lktC::cat chromosomal fusions, to study transcription initiation in M. haemolytica. Using these genetic tools, we have identified cis-acting sequences and environmental conditions that modulate transcription of the leukotoxin and lapT promoters. By deletion analysis, promoters were shown to rely on sequences upstream of their -10 and -35 regions for full activity. Direct repeats of the sequence TGT-N(11)-ACA and a static bend region caused by phased adenine tracts were necessary for full activation of P(lkt). A computer-generated model of the promoter's structure shows how DNA bending brings the repeat sequences within close proximity to the P(lkt) RNA polymerase, and we hypothesize that these repeats are a binding site for an activator of leukotoxin transcription. The lktC::cat operon fusion was also used to demonstrate that, like that of other RTX toxins, leukotoxin transcription is environmentally regulated. Roles for iron deprivation and temperature change were identified.

Bordetella bronchiseptica fimbrial protein-enhanced immunogenicity of a Mannheimia haemolytica leukotoxin fragment

Leukotoxin produced by Mannheimia ( Pasteurella) haemolytica is an important virulence factor in shipping fever pneumonia in feedlot cattle and is a critical protective antigen. In this study, the immune response to a chimeric protein generated by combining a gene fragment encoding neutralizing epitopes of M. haemolytica leukotoxin and a fimbrial protein gene ( fim N) from Bordetella bronchiseptica was evaluated. The recombinant gene was cloned in a bacterial expression vector under the control of the tac promoter and expressed as a fusion protein with glutathione- S-transferase (GST) in Escherichia coli. Immunization of mice with the recombinant protein, GST-LTXFIM elicited a significantly stronger anti-leukotoxin antibody response than comparable immunizations with GST-LTX fusion proteins lacking FIM N. The GST-LTXFIM was also more stable than GST-LTX during storage at −80 °C, thus alleviating a stability problem inherent to leukotoxin. This chimeric protein may be a candidate for inclusion in new generation vaccines against shipping fever pneumonia.

Infectivity-neutralizing and hemagglutinin-inhibiting antibody responses to respiratory coronavirus infections of cattle in pathogenesis of shipping fever pneumonia.

Respiratory bovine coronaviruses (RBCV) emerged as an infectious agent most frequently isolated from respiratory tract samples of cattle with acute respiratory tract diseases. Infectivity-neutralizing (IN) and hemagglutinin-inhibiting (HAI) antibodies induced by RBCV infections were monitored in sequential serum samples collected from cattle during a naturally evolving and experimentally monitored epizootic of shipping fever pneumonia (SFP). Cattle nasally shedding RBCV at the beginning of the epizootic started with low levels of serum IN and HAI antibodies. An increase in serum IN antibody after day 7 led to reduction of virus shedding in nasal secretions by the majority of the cattle between days 7 and 14. A substantial rise in the serum HAI antibody was observed during the initial phase among the sick but not the clinically normal cattle which were infected with RBCV. The RBCV isolation-positive cattle that developed fatal SFP had minimal serum IN and HAI antibodies during the course of disease development. Cattle that remained negative in RBCV isolation tests entered this epizootic with high levels of serum IN and HAI antibodies, which dramatically increased during the next two weeks. Protection against SFP was apparently associated with significantly higher levels of serum IN antibodies at the beginning of the epizootic. The RBCV-neutralizing activity is associated with serum immunoglobulin G (IgG), particularly the IgG2 subclass, while RBCV-specific HAI antibody is related to both serum IgG and IgM fractions.

Molecular genetic analysis of virulence in Mannheimia (pasteurella) haemolytica.

Mannheimia haemolytica (previously known as Pasteurella haemolytica) is a weakly hemolytic, gram-negative coccobacillus that is an opportunistic pathogen of cattle, sheep and other ruminants. In stressed, immunocompromised animals, the organism causes a fibrinous, necrotic pneumonia, commonly called "shipping fever". In the United States, economic losses due to shipping fever pneumonia surpass the combined cost of all other diseases of cattle. M. haemolytica, which is a member of the family Pasteurelleaceae, includes twelve serotypes (A1, A2, A5-A9, A12-14, A16 and A17) based on capsular antigen typing. Worldwide, serotypes A1 and A2 predominate, though all serotypes can cause disease. Serotype A1 causes pasteurellosis in cattle and has been the subject extensive study, while serotype A2 causes disease in sheep and is less-well characterized. Potential virulence factors of M. haemolytica have been identified and characterized by gene cloning and DNA sequence analysis. These factors include a ruminant-specific leukotoxin, an anti-phagocytic capsule, lipopolysaccharide, iron-regulated outer membrane proteins, lipoproteins, a sialoglycoprotease, a neuraminidase and two potential immunoglobulin proteases. Unlike the well-characterized leukotoxin, little is known about the expression of these other virulence factors. Attempts to dissect the mechanisms of M. haemolytica pathogenesis have been hindered by the lack of a robust genetic system for mutation of the organism, though new tools for genetic manipulation of M. haemolytica have been developed. Expression plasmids and operon fusion plasmids have been created and a series of antibiotic resistance cassettes useful for site-specific recombination have been constructed. It is anticipated that use of these tools for gene expression and mutagenesis, in combination with the soon to be released genomic sequence of a serotype A1 organism, will aid in understanding the molecular mechanisms of pathogenesis of M. haemolytica and will help to drive development of new vaccines to prevent shipping fever.

Coronavirus and Pasteurella infections in bovine shipping fever pneumonia and Evans' criteria for causation.

Respiratory tract infections with viruses and Pasteurella spp. were determined sequentially among 26 cattle that died during two severe epizootics of shipping fever pneumonia. Nasal swab and serum samples were collected prior to onset of the epizootics, during disease progression, and after death, when necropsies were performed and lung samples were collected. Eighteen normal control cattle also were sampled at the beginning of the epizootics as well as at weekly intervals for 4 weeks. Respiratory bovine coronaviruses (RBCV) were isolated from nasal secretions of 21 and 25 cattle before and after transport. Two and 17 cattle nasally shed Pasteurella spp. before and after transport, respectively. RBCV were isolated at titers of 1 x 10(3) to 1.2 x 10(7) PFU per g of lung tissue from 18 cattle that died within 7 days of the epizootics, but not from the lungs of the remaining cattle that died on days 9 to 36. Twenty-five of the 26 lung samples were positive for Pasteurella spp., and their CFU ranged between 4.0 x 10(5) and 2.3 x 10(9) per g. Acute and subacute exudative, necrotizing lobar pneumonia characterized the lung lesions of these cattle with a majority of pneumonic lung lobes exhibiting fibronecrotic and exudative changes typical of pneumonic pasteurellosis, but other lung lobules had histological changes consisting of bronchiolitis and alveolitis typical of virus-induced changes. These cattle were immunologically naive to both infectious agents at the onset of the epizootics, but those that died after day 7 had rising antibody titers against RBCV and Pasteurella haemolytica. In contrast, the 18 clinically normal and RBCV isolation-negative cattle had high hemagglutinin inhibition antibody titers to RBCV from the beginning, while their antibody responses to P. haemolytica antigens were delayed. Evans' criteria for causation were applied to our findings because of the multifactorial nature of shipping fever pneumonia. This analysis identified RBCV as the primary inciting cause in these two epizootics. These viruses were previously not recognized as a causative agent in this complex respiratory tract disease of cattle.

Correlation of Pasteurella haemolytica leukotoxin binding with susceptibility to intoxication of lymphoid cells from various species.

Pasteurella haemolytica, the causative agent of shipping fever pneumonia in cattle, produces a leukotoxin (LKT) which lyses ruminant leukocytes with high efficiency but is reputed to not affect leukocytes from nonruminant species. In this study, we tested the supposition that LKT binding correlates positively with susceptibility to intoxication of susceptible isolated bovine lymphocytes and lymphoma tissue culture cells (BL3 cells) and negatively with reputed nonsusceptible equine, porcine, and canine lymphocytes and human lymphoid tissue culture cells (Raji cells). Bovine lymphocytes and BL3 cells were highly susceptible to LKT intoxication, exhibiting both substantial increase in intracellular Ca(2+) concentration and marked leukolysis. Exposure of reputed LKT-nonsusceptible porcine lymphocytes and Raji cells to LKT caused a slightly increased intracellular Ca(2+) concentration but no leukolysis. No LKT effect was detected for equine and canine lymphocytes. LKT bound to lymphoid cells from all species tested. Intact 102-kDa LKT was recovered from exposed isolated lymphoid cell membranes. Pro-LKT acylation was not required for LKT binding to BL3 cells. LKT binding was rapid, with maximal binding occurring by 3 min, and was proportional to the LKT concentration in the range 0.04 to 4.0 microg/ml. For this LKT concentration range, BL3 cells bound more LKT than did porcine lymphocytes or Raji cells, suggesting that LKT binds to BL3 cells with higher affinity than to porcine lymphocytes or Raji cells. Above 4.0 microg/ml, LKT demonstrated saturable binding to BL3 cells. Neutralizing anti-LKT monoclonal antibody (MAb) MM601 diminished LKT binding to BL3 by 36% while decreasing leukolysis by 81%. In contrast, MM601 did not diminish LKT binding to Raji cells. Pretreatment of target cells with 120 microg of protease K per ml diminished LKT binding to BL3 cells by 75%, with only a 25% decrease in leukolysis. However, pretreatment with 150 microg of protease K per ml abolished the remaining 25% of LKT binding and 75% leukolysis. Therefore, P. haemolytica LKT binds rapidly to susceptible and to reputed nonsusceptible lymphoid cells. LKT binding resulting in species-specific leukolysis was characterized by high affinity, inhibition by MAb MM601, and relative resistance to protease K pretreatment of lymphoid cells. Two types of LKT binding to lymphoid cells are proposed. High-affinity binding leads to efficient leukolysis. In some lymphoid cells from reputed LKT-nonsusceptible species, low-affinity LKT binding may cause a low-efficiency increase in the intracellular Ca(2+) concentration without leading to leukolysis.

Identification of immunogenic, surface-exposed outer membrane proteins of Pasteurella haemolytica serotype 1

Pasteurella haemolytica serotype 1 (S1) is the bacterium most frequently recovered from the lungs of cattle that have succumbed to shipping fever pneumonia. P. haemolytica outer membrane proteins (OMPs) are important immunogens in the development of resistance to pneumonic pasteurellosis. The purpose of this study was to identify the repertoire of immunogenic, surface-exposed P. haemolytica (S1) OMPs, that could be important in the development of protective immunity. We determined surface exposure of OMPs by (1) their susceptibility to protease treatment and (2) their ability to adsorb out antibodies from bovine immune sera. For a comprehensive identification of immunogenic, surface-exposed OMPs, we used bovine antisera from calves that were resistant to experimental P. haemolytica challenge after (1) natural exposure to P. haemolytica, (2) vaccination with live P. haemolytica, or (3) vaccination with P. haemolytica OMPs. We identified 21 immunogenic, surface-exposed P. haemolytica OMPs. Most were recognized by all three immune sera. However, some were recognized by one or two of the three antisera. Our analyses identified surface-exposed, immunogenic proteins that were not identified in previous studies.

Restriction endonuclease analysis and ribotyping differentiate Pasteurella haemolytica serotype A1 isolates from cattle within a feedlot.

Pasteurella haemolytica serotype A1 isolates were collected from cattle within a feedlot during an outbreak of bovine respiratory disease. Genetic heterogeneity among the isolates was examined by restriction endonuclease analysis (REA), ribotyping, and analysis of plasmid content. The susceptibilities of isolates to several antibiotics were also examined. Five different REA patterns and three different ribotypes were observed among the isolates. Fifty percent of the isolates had an identical REA type, ribotype, and plasmid profile. Examination of the plasmid content of the isolates revealed that most (73%) carry a single plasmid which encodes beta-lactamase, 13.5% carry two plasmids, and 13.5% carry no plasmid. The data reveal the presence of genetic differences among isolates of P. haemolytica A1, associated with shipping fever pneumonia within a closed feedlot, and suggest that a combination of REA, ribotyping, plasmid analysis, and antibiotic susceptibility determination will be useful in analyzing the molecular epidemiology of this disease.

A novel dialysis fermentor design and its application to the production of a cytotoxin fromPasteurella haemolytica

A multi-compartment dialysis membrane fermentor, with a combined compartment volume of one liter, was designed with vertical dialysis bag placement and individual bag inoculation ports. These ports, based on an adaptation of standard SwagelokR bulkhead type stainless steel fittings, allowed sterile access to each bag lumen during growth. This design has been used extensively to study the growth characteristics and secreted macromolecular products fromPasteurella haemolytica cultures isolated from bovine lung infected with shipping fever pneumonia.

Blood Chemistry Weaning Stress Study

Shipping fever complex in the weaning calf is a paramount problem to the beef cattle industry. The highest incidence of disease and death loss is associated with weaning, transportation, handling, and ration adaptation. Most research has concentrated on the bacterial and viral aspects; however, some research has been done on the changes in physiological status of the animal as a result of stress. These changes are related to clinical signs, blood composition alterations, and adrenal gland changes (1). Perhaps the most constant indicator of stress has been the changes in the activity of the adrenal gland. The cortisol activities on the immune system in the weaned and trucked calf may play a major role in the susceptibility and subsequent disease in this calf. This study was conducted to evaluate the effect of weaning stress on 10 blood serum components, hematology, and weight changes. As in the previous study, the most noticeable changes were in the cortisol level and weight change (1). Our general observation on this study and a similar study conducted two years ago is as follows: Animals weaned 4 weeks prior to trucking undergo a stress period lasting 3 to 7 days. Weaning alone stresses these animals for about the same period. Concurrent weaning and trucking increases the stress period to 15-21 days. These conclusions are based upon changes observed in the cortisol, LDM, SGOT, WBC and weight change. The majority of the shipping fever pneumonia occurs in the first 45 days following weaning, caused by the interaction of stresses, viruses, and bacteria (2,3). In this study, we were able to show that the weaning and trucking produced the most stress and failure to gain weight. The higher cortisol the first 7 days and not returning to normal for 15-21 days may suppress the immune system of the calf. An interesting follow-up of this study would be to evaluate the responses to a known antigen in each of these groups.