Tissue-specific expression of the rat alpha 2u globulin gene family.

The rat alpha 2u globulin gene family encodes approximately 20 low-molecular-weight (20,000) proteins with pIs ranging from 4.5 to 7.9. alpha 2u globulin protein isoforms were detected in the liver and in the submaxillary, lachrymal, preputial, and mammary glands of Sprague-Dawley rats. The hormonal and developmental regulation of alpha 2u globulin synthesis in each of these tissues was unique, and it appears that different alpha 2u gene sets were transcribed in the various tissues.

Comparison of biologic behavior and histology of transplanted mammary tumors in GR mice.

Mammary tumors that arose spontaneously in inbred GR mice were transplanted into syngeneic castrated males. The hormone responsiveness of the transplants was studied in mice treated with estrone and progesterone and was compared with the hormone responsiveness in mice that received no hormone treatment. Microscopic examination of hormone-responsive and hormone-independent tumors revealed similar histologic patterns in both groups. It was evident that pale cells, which are classically associated with hormone-responsive tumors, may also be present in transplanted hormone-independent tumors in this strain. No correlation was found between the histologic pattern of these transplanted tumors and the biologic behavior, hormonal status, or presence of a specific murine mammary tumor virus (MuMTV) proviral fragment. Mammary tumors also appeared capable of undergoing differentiation into more than one morphologic type. Two cotransplanted tumors (derived from the same parental tumor) had markedly different histologic patterns; however, analysis of MuMTV proviral fragments indicated that the MuMTV-infected cells were of the same parentage.

Purification of HlyX, a potential regulator of haemolysin synthesis, and properties of HlyX:FNR hybrids.

The hlyX gene of the swine pathogen Actinobacillus pleuropneumoniae is homologous to FNR, an anaerobic transcriptional regulator of Escherichia coli. It endows a haemolytic phenotype upon E. coli, and will complement the anaerobic respiratory deficiencies of fnr mutants of E. coli. The coding region of the hlyX gene was expressed in E. coli and the HlyX protein was purified by using an assay based on its immunological cross-reactivity with anti-FNR antibodies. The HlyX protein had the predicted N-terminal sequence, and resembled the isolated FNR protein in size (Mr 29,000) and monomeric organization. It has no detectable haemolysin activity per se, and is therefore presumed to confer a haemolytic phenotype by activating a latent haemolysin gene in E. coli. Studies with gene fusions showed that HlyX, like FNR, can function as an anaerobic activator and repressor of FNR-regulated genes in vivo. Plasmids that express hybrid HlyX:FNR proteins in which the 189/190-residue N-terminal segments and the remaining 50/60-residue C-terminal segments are exchanged, retained their FNR-specific functions but failed to confer a haemolytic phenotype. This suggests that the specificity for activating the haemolytic response requires the participation of unique features in both the N- and C-terminal segments of HlyX.

Expression and phylogenetic relationships of a novel lacZ homologue from Actinobacillus pleuropneumoniae.

To learn more about the genetics and physiology of the important swine pathogen, Actinobacillus pleuropneumoniae, we cloned the lacZ gene by complementation of an Escherichia coli delta lac mutant. The A. pleuropneumoniae lacZ gene has an open reading frame of 3015 bp which could encode a protein with a predicted molecular mass of 117022. The deduced protein shares 26.8-34.8% identity with beta-galactosidases from both Gram-positive and Gram-negative bacteria. Sequences with homology to seven regions commonly found in beta-galactosidases are present and amino acids corresponding to active site residues Tyr-503 and Glu-537 in E. coli LacZ are also conserved; however, there is a leucine in the place of Gly-794, a residue which has been implicated in substrate recognition. The sequences flanking the A. pleuropneumoniae lacZ gene do not share homology with known transport or regulatory genes nor do they share homology with cAMP receptor protein (CRP) or LacI binding sites. Low levels of beta-galactosidase activity could be detected when the protein was expressed from a multicopy plasmid in E. coli delta lac and when it was measured in A. pleuropneumoniae. The level of activity was not markedly reduced in the presence of glucose. Although the A. pleuropneumoniae LacZ shares some features with other beta-galactosidases, its constitutive expression and an unusual active site residue suggest that it may have a unique function.

A search for virulence genes of Haemophilus parasuis using differential display RT-PCR.

Although Haemophilus parasuis is an important bacterial pathogen of swine, little is known about its pathogenesis or why some strains seem to be more virulent than others. Therefore, we used differential display reverse transcription-polymerase chain reaction (DDRT-PCR) to search for virulence-associated genes in a pathogenic serotype 5 strain, H. parasuis 1185. Gene expression was evaluated following growth in conditions chosen to begin to approximate those found in the upper respiratory tract and those encountered by the organism during acute infection. Seven different differentially expressed gene fragments were identified in cells grown at 40 degrees C in both the presence and absence of swine serum. Based on the deduced amino acid sequences, the most strongly up-regulated genes were homologs of fadD (a fatty acyl-CoA synthetase), apaH (diadenosine tetraphosphatase), pstI (enzyme I of the phosphoenolpyruvate-protein phosphotransferase system), and cysK (cysteine synthetase). Homologs of Std (Na(+)- and Cl(-)-dependent ion transporter), HSPG (a mammalian basement membrane-specific heparin sulphate core protein precursor) and PntB (pyridine nucleotide transhydrogenase) were also up-regulated, but to a much lower extent. Sequences homologous to all of the differentially expressed genes were detected in the reference strains of all 15 H. parasuis serotypes. This is the first report of a global search for virulence factors of H. parasuis.

The first description of a (1--> 6)-beta-D-glucan in prokaryotes: (1 --> 6)-beta-D-glucan is a common component of actinobacillus suis and is the basis for a serotyping system.

The chemical and antigenic properties of the cell-surface lipopolysaccharides (LPSs) and capsular polysaccharides (CPSs) of seven representative strains of Actinobacillus suis from healthy and diseased pigs were investigated. Four strains produced a linear (1 --> 6)-beta-D-glucan homopolymer, beta-D-Glcp-(1-[ --> 6)-beta-D-Glcp-(1-]n -->, as a LPS-O-chain (O1) and as a CPS (K1). Polyclonal antisera prepared against a (1 --> 6)-beta-D-glucan-containing strain showed a positive reaction against both LPSs and CPSs derived from the above strains (designated serotype O1/K1). One strain carried the (1 --> 6)-beta-D-glucan solely as a LPS-O-chain (serotype O1) and two strains did not express the (1 --> 6)-beta-D-glucan, but, instead, produced a different O-chain (designated serotype 02); these three strains expressed their own characteristic CPSs. (1 --> 6)-beta-D-Glucan structures are common cell wall components of yeast, fungi and lichens, but, to our knowledge, this is the first time a (1 --> 6)-beta-D-glucan has been described in a prokaryotic organism. Conformational and nuclear magnetic resonance analyses showed that the beta-D-Glcp-(1 --> 6)-beta-D-Glcp linkage was flexible and two distinct glycosidic conformers are described. Cross-reactive antibodies to the A. suis (1 --> 6)-beta-D-glucan could be detected in sera from a variety of species and in sera from specific pathogen free pigs. This cross-reactivity may arise from immuno-stimulation of organisms present in the surrounding environment that contain (1 --> 6)-beta-D-glucan, which may also explain the high incidence of false positive results in previous serological tests for A. suis. In addition, these (1 --> 6)-beta-D-glucan background antibodies may be protective against A. suis infection. The characterization herein of (1 --> 6)-beta-D-glucan is the foundation for the development of a serotyping system for A. suis.

The family Pasteurellaceae: modern approaches to taxonomy.

Historically, members of the family Pasteurellaceae were classified on the basis of a limited number of phenotypic characteristics. In particular, organisms were assigned to the family on the basis of their requirements for the growth factors hemin and/or nicotinamide adenine dinucleotide and on the basis of their ability to cause disease in vertebrates. More recent genotypic studies have shown that many members of the family Pasteurellaceae have been misclassified. In this paper we review some of the current taxonomic methods which can be used to emend the classification of Haemophilus, Actinobacillus and Pasteurella spp. and suggest some approaches and criteria for restructuring the family Pasteurellaceae.

Urease activity may contribute to the ability of Actinobacillus pleuropneumoniae to establish infection.

The contribution of urease activity to the pathogenesis of Actinobacillus pleuropneumoniae was investigated using 2 different urease-negative transposon mutants of the virulent serotype 1 strain, CM5 Nalr. One mutant, cbiK::Tn10, is deficient in the uptake of nickel, a cofactor required for urease activity. The other mutant, ureG::Tn10, is unable to produce active urease due to mutation of the urease accessory gene, ureG. In aerosol challenge experiments, pigs developed acute pleuropneumonia following exposure to high doses (10(6) cfu/mL) of the parental strain, CM5 Nalr, and to the cbiK::Tn10 mutant. When low dose (10(3) cfu/mL) challenges were used, neither urease-negative mutant was able to establish infection, whereas the parental strain was able to colonize and cause lesions consistent with acute pleuropneumonia in 8 of the 20 pigs challenged. These findings suggest that urease activity may be needed for A. pleuropneumoniae to establish infection in the respiratory tract of pigs.

Agents of the "suis-ide diseases" of swine: Actinobacillus suis, Haemophilus parasuis, and Streptococcus suis.

In recent years, Actinobacillus suis, Haemophilus parasuis, and Streptococcus suis have emerged as important pathogens of swine, particularly in high health status herds. Their association with a wide range of serious clinical conditions and has given rise to the moniker "suis-ide diseases." These organisms are early colonizers and, for that reason, are difficult to control by management procedures such as segregated early weaning. Vaccination, serodiagnostic testing, and even serotyping are complicated by the presence of multiple serotypes, cross-reactive antigens, and the absence of clear markers for virulence. In this review, we discuss our current understanding of the pathogenesis, epidemiology, and management of the causative agents of the "suis-ide diseases" of swine.

Analysis of Haemophilus parasuis isolates from southern Ontario swine by restriction endonuclease fingerprinting.

To study the occurrence and distribution of various strains of Haemophilus parasuis in southern Ontario swine, organisms isolated from healthy swine, from specific pathogen-free and conventional herds, and from disease cases were examined using restriction endonuclease fingerprinting analysis. In most herds, several strains of H. parasuis could be detected although one or two strains usually predominated. Individual animals were colonized by a single or limited number of strains. In several cases, the same strains were isolated from more than one specific pathogen-free herd. Conventional herds carried a more heterogeneous population of H. parasuis. Only one strain was isolated which was common to more than one conventional herd. No strains were isolated which were common to both specific pathogen-free and conventional herds. None of the strains isolated from disease cases were found in healthy conventional or specific pathogen-free swine examined in this study.

Analysis of southern Ontario Actinobacillus (Haemophilus) pleuropneumoniae isolates by restriction endonuclease fingerprinting.

Isolates of Actinobacillus (Haemophilus) pleuropheumoniae were studied by restriction endonuclease fingerprinting (REF) analysis using the enzymes BamHI and HindIII. Restriction fragments were resolved by polyacrylamide gel electrophoresis and visualized by silver staining. Except for serotypes 1 and 9, reference strains of A. pleuropneumoniae serotypes 1 to 10 had clearly distinguishable REF profiles. Analysis of REF profiles of southern Ontario field isolates revealed limited heterogeneity amongst isolates of serotype 1 or serotype 5. The REF profiles of the serotype 7 isolates studied showed greater variation. Heterogeneity could not be correlated with the presence of plasmids nor with antibiotic resistance. Limited heterogeneity could also be detected amongst REF profiles of A. pleuropneumoniae isolates recovered from a closed herd suggesting that there is a small amount of genetic variation within clonal populations.

Comparative pathogenicity of different Actinobacillus suis O/K serotypes.

The pathogenicity of Actinobacillus suis serotypes O1/K1 (strain SO4), O1/K2 (strain C84), and O2/K2 (strain H91-0380) was evaluated in specific-pathogen-free (SPF) piglets challenged by intraperitoneal inoculation with approximately 1 x 10(7) colony-forming units per mL. All 3 strains produced peritonitis, but differences were observed in the composite histopathologic scores (P = 0.001) and in their ability to spread (P = 0.008) at 7 h post challenge. The O2/K2 strain caused the most severe peritonitis and disseminated most widely to other tissues. Moderate lesions were seen with the O1/K2 strain while the O1/K1 strain caused mild lesions and remained largely localized to the peritoneum. In an attempt to explain the basis of observed differences, the serum sensitivity of 9 A. suis strains with different O and K types was assessed. Regardless of the O/K type, all of the isolates tested were serum resistant. Moreover, most A. suis isolates grew as well or better in complement-replete sera as they did in complement-depleted sera. These observations indicate that although 02 and K2 strains had a greater propensity to cause a disseminating septic inflammatory response in pigs, they were no more resistant to complement-mediated killing than O1 strains.

Experimental sialodacryoadenitis virus infection in athymic CD-1 mice.

The purpose of the study was to investigate the susceptibility of nude mice to sialodacryoadenitis virus. Young adult male CD-1 nude mice were inoculated intranasally with virus, killed at 2, 4, 6, 8, 10 and 20 days postinoculation and examined for virus-induced lesions in tissues including respiratory tract. Inoculated and control mice were examined by virus isolation and serology. In a companion study, male Wistar rats were inoculated intranasally with the same inoculum, and examined by histopathology, immunofluorescence microscopy and serology. In virus-inoculated mice, lesions were minimal in the lower respiratory tract, and were absent in other tissues. Virus was isolated from the lower respiratory tract in animals sampled at six or eight days postinoculation. Antiviral antibody was not detected in sera from inoculated and control mice. Virus-associated lesions and antibodies were readily detected in rats following inoculation. Based on this study, there is no evidence that inadvertent exposure to sialodacryoadenitis virus should pose a threat to CD-1 nude mice, and their susceptibility to the disease appears to be similar to that reported in euthymic CD-1 mice.

Preliminary characterization of the structural proteins of the coronaviruses, sialodacryoadenitis virus and Parker's rat coronavirus.

A procedure was developed for the partial purification of the rat coronaviruses, sialodacryoadenitis virus (SDAV) and Parker's rat coronavirus (PRC). The SDAV and PRC were replicated in L-2 cell monolayer cultures, precipitated with ammonium sulphate, and further concentrated using sucrose density gradient centrifugation. The major SDAV and PRC proteins were identified by immunoblotting and compared with those of the JHM strain of mouse hepatitis virus (MHV-JHM). Monoclonal antibodies (MAb) against the M protein of JHM recognized proteins interpreted to be slightly smaller in immunoblots of SDAV and PRC (22.8 vs 23K for JHM). Similarly, a monoclonal antibody against the JHM N protein reacted with proteins of 53K in SDAV and PRC (vs 56 K for JHM). Polyclonal antisera to all three viruses also cross-reacted with the M and N proteins. Some cross-reactivity amongst the S proteins was observed. Based on these data, the structural proteins of the rat coronaviruses, SDAV and PRC are closely related to those of MHV-JHM.

Characterization of an attenuated strain of Actinobacillus pleuropneumoniae, serotype 1.

Pleuropneumonia is an important disease of swine caused by Actinobacillus pleuropneumoniae. Putative virulence determinants include capsule, lipopolysaccharide, and cytotoxin. We studied the virulence and virulence determinants of 2 strains: CM5 and CM5A of serotype 1. Strain CM5 was isolated from a pig with pleuropneumonia and passaged once in vitro; strain CM5A was a substrain of CM5 passaged 70 times in vitro. Pigs challenge exposed to an aerosol of 1.3 x 10(7) colony-forming units of CM5/ml died within 30 hours; pigs challenge exposed to an aerosol of 1.6 x 10(8) colony-forming units of CM5A/ml survived. The average thickness of the capsular layer was 137 nm in strain CM5 and 53 nm in strain CM5A in bacteria treated with homologous antibody and examined by transmission electron microscopy. Similarly, capsular material binding polycationic ferritin was found in colonies of strain CM5, but not in strain CM5A. The ratio of hexosamine to protein in extracted capsule of CM5 was more than twice that of CM5A. The polyacrylamide gel electrophoretic profile of the lipopolysaccharide, outer membrane proteins, and whole cell proteins did not differ between the 2 strains. Also, the amount of cytotoxin or endotoxin produced by the 2 strains during the logarithmic growth phase was not different. The electrophoretic profile of restriction endonuclease digested DNA was similar, with the exception of bands in the 750- and 620-basepair regions. It was concluded that attenuation of strain CM5A during in vitro passage was a result of reduced capsule production and that encapsulation is an important virulence determinant of A pleuropneumoniae, serotype 1.

Evaluation of heat-sensitive, neutrophil-toxic, and hemolytic activity of Haemophilus (Actinobacillus) pleuropneumoniae.

Cytotoxic and hemolytic activity of Haemophilus (Actinobacillus) pleuropneumoniae serotype 1 strain CM5 was investigated because of the potential role as a virulence determinant. Viable bacteria were toxic for porcine and bovine neutrophils, whereas bacteria killed by heat treatment at 60 C for 1 hour were not. Similarly, bacteria-free culture supernatant was cytotoxic and hemolytic in assays that used porcine neutrophils and erythrocytes, whereas supernatant treated at 60 C for 1 hour had no activity. Erythrocytes from various species were susceptible to the hemolytic activity of bacteria-free culture supernatant, with ovine and bovine erythrocytes being most sensitive. The neutrophil-toxic and hemolytic activity of bacteria-free culture supernatant was inhibited by cholesterol and oxygen and abolished after trypsin digestion. The neutrophil-toxic and hemolytic activity was preserved during storage at or less than 4 C, but was lost rapidly at 56 C or 80 C. Neutralizing antibodies were demonstrated in serum of pigs and rabbits immunized with 10-fold concentrated culture supernatant of strain CM5 and in field pigs that had recovered from natural infection with H pleuropneumoniae serotype 1. Bacteria-free culture supernatants of 18 strains, including H pleuropneumoniae serotypes 1 through 10, Actinobacillus suis, and Haemophilus taxon minor group, were tested for heat-sensitive, neutrophil-toxic, and hemolytic activity. Fifteen strains were neutrophil toxic, but only 10 of these were hemolytic. Haemophilus pleuropneumoniae, serotype 1, strain VLS557; serotype 5, strain K17; and Haemophilus taxon minor group strain 33PN were neither cytotoxic nor hemolytic.

An investigation of enzootic Glasser's disease in a specific-pathogen-free grower-finisher facility using restriction endonuclease analysis.

Enzootic Glassers's disease was investigated to study the epidemiology of the disease strains on a farm where it presented a problem. Restriction endonuclease fingerprinting (REF) analysis technique was used, as all strains of Haemophilus parasuis are biochemically similar and many strains are biochemically untypable. After young weaned pigs were moved from farm A to farm B, Glasser's disease routinely occurred despite the use of antibiotics and a commercial bacterin. Isolates were taken from the nasal passages and from carcasses of clinically affected cases and subjected to REF analysis. Haemophilus parasuis was not isolated from any of the pigs on farm A, but it was isolated from 7/10 and 5/10 nasal swabs taken from farm B. Two H. parasuis strains isolated from clinical cases of Glasser's disease from farm B had an identical REF pattern, but were different from the nasal swabs and the H. parasuis strain contained in the bacterin. The subsequent use of a custom autogenous bacterin made from a clinical isolate of H. parasuis reduced the mortality rate on farm B. This investigation indicates that nasal isolates of H. parasuis are different than those causing clinical disease, and not all bacterin strains are cross protective for other strains.

Characterization of colonization-deficient mutants of Actinobacillus suis.

Actinobacillus suis is an important opportunistic pathogen of swine that can cause disease in pigs of all ages, especially in high-health status herds. Although A. suis shares many virulence factors in common with Actinobacillus pleuropneumoniae and can cause a haemorrhagic pleuropneumonia similar to that caused by A. pleuropneumoniae, A. suis most often causes septicaemia and diseases such as arthritis and meningitis that are sequelae to septicaemia. In a recent signature-tagged transposon mutagenesis study, 30 colonization-essential genes of A. suis were identified. In the current study, the attachment and invasion patterns of strains harboring Tn10 insertions in ompA, pfhaB1, lcbB, and cpxR were evaluated using porcine palatine tonsil organ cultures, the swine kidney epithelial cell line, SK6, and a porcine brain microvascular endothelial cell line, PBMEC/C1-2. All of these mutants attached in lower numbers than wild type to the tonsillar explants and to the SK6 cells. The ompA mutant attached in significantly lower numbers than wild type to the porcine tonsil cells (P=0.02) and to PBMEC (P=0.0008) at 60 min time point. As well, the ompA mutant showed significantly greater sensitivity than wild type to chemical stressors and to swine serum. Using fluorescent microscopy, a GST-OmpA fusion protein could be demonstrated to interact with the crypt epithelial cells of porcine palatine tonsil.

Genetic and biochemical analyses of Actinobacillus pleuropneumoniae urease.

The urease gene cluster from the virulent Actinobacillus pleuropneumoniae serotype 1 strain CM5 was cloned and sequenced. The urease activity was associated with a 6.3-kbp region which contains eight long open reading frames (ORFs). The structural genes, ureABC, are separated from the accessory genes, ureEFGD, by a 615-bp ORF of unknown function, ureX. Homologies were found with the structural and accessory urease gene products of Haemophilus influenzae and, to a lesser extent, with those of other organisms. The urease enzyme subunits had predicted molecular masses of 61.0, 11.3, and 11.0 kDa, and the size of the holoenzyme was estimated to be 337 +/- 13 kDa by gel filtration chromatography. Urease activity was maximal but unstable at 65 degrees C. In cell lysates, the A. pleuropneumoniae urease was stable over a broad pH range (5.0 to 10.6) and the optimal pH for activity was 7.7. The Km was 1.5 +/- 0.1 mM urea when it was assayed at pH 7.7. The low Km suggests that this enzyme would be active in the respiratory tract environment, where urea levels should be similar to those normally found in pig serum (2 to 7 mM).