Search

Moraxella bovis is the causative agent of infectious bovine keratoconjunctivitis (IBK) also known as pinkeye, a highly contagious and painful eye disease that is common in cattle throughout the world. Vaccination appears to be a reasonable and cost-effective means of control of pinkeye. Identification of genes encoding novel secreted antigens have been reported, and these antigens are being assessed for use in a vaccine. One of the genes encodes phospholipase B, which can be expressed with high purity and yield in recombinant Escherichia coli as a secreted, soluble, non-tagged, mature construct (less signal peptide with predicted mass 63 kDa). The recombinant phospholipase B exhibited anomalous electrophoretic mobility that was dependent on the temperature of the denaturing process, with bands observed at either 52 or 63 kDa. Analysis by in-gel digestion and liquid chromatography-mass spectrometry revealed these two distinct forms most likely had identical sequences. Phospholipase B is a compact, globular protein with a predicted structure typical of a conventional autotransporter. It is suggested that high temperature is required to unfold the protein (to denature the beta-barrel-rich transporter domain) and to ensure accessibility of the reducing agent. Interestingly, the two forms of the enzyme, differing in size and isoelectric points, were also detected in cell-free supernatants of M. bovis cultures, indicating that native phospholipase B may exist in two differentially folded states possibly also differing in oxidation status of cysteine residues.

Menangle virus (MenV), which was isolated in Australia in 1997 during an outbreak of severe reproductive disease in pigs, is a novel member of the genus Rubulavirus in the family Paramyxoviridae. Although successfully eradicated from the affected piggery, fruit bats are considered to be the natural reservoir of the virus and therefore an ongoing risk of re-introduction to the pig population exists. Accordingly, reagents to facilitate serological surveillance are required to enhance the diagnostic capability for MenV, which is a newly recognized cause of disease in pigs with the potential to severely affect production in naive breeding herds. To address this need, recombinant MenV nucleocapsid (N) protein was expressed in the yeast Saccharomyces cerevisiae. Using the expression vector pFGG3 under control of the GAL7 promoter, high yields of recombinant MenV N protein were obtained. Electron microscopy demonstrated that purified recombinant N protein self-assembled into nucleocapsid-like particles which were identical in density and morphology, although not in length, to authentic nucleocapsids from virus-infected cells. Electron microscopy analysis also showed that yeast-expressed N protein which lacked the C-terminal tail (amino acid residues 400-519) formed significantly longer and denser nucleocapsid-like particles. Nucleocapsid-like particles derived from the full-length recombinant protein were stable and readily purified by CsCl gradient ultracentrifugation. When used as coating antigen in an indirect ELISA, the recombinant N protein reacted with sera derived from pigs experimentally infected with MenV and a simple serological assay to detect MenV-specific antibodies in pigs, fruit bats and humans could be designed on this basis.

The aims were to longitudinally evaluate the interferon-gamma (IFN-gamma) test in comparison to faecal culture and the absorbed ELISA in a cattle infection model for Johne's disease and to determine the adult infection status, by necropsy and tissue culture, of sheep, goats and cattle infected as young animals. Clinical disease, faecal culture results and immunological responses for Merino sheep [Stewart, D.J., Vaughan, J.A., Stiles, P.L., Noske, P.J., Tizard, M.L.V., Prowse, S.J., Michalski, W.P., Butler, K.L., Jones, S.L., 2004. A long-term study in Merino sheep experimentally infected with Mycobacterium avium subsp. paratuberculosis: clinical disease, faecal culture and immunological studies. Vet. Microbiol. 104, 165-178] and Angora goats [Stewart, D.J., Vaughan, J.A., Stiles, P.L., Noske, P.J., Tizard, M.L.V., Prowse, S.J., Michalski, W.P., Butler, K.L., Jones, S.L., 2006. A long-term study in Angora goats experimentally infected with Mycobacterium avium subsp. paratuberculosis: clinical disease, faecal culture and immunological studies. Vet. Microbiol. 113, 13-24], in the same experiments as the Holstein-Friesian cattle, have been described. Two longitudinal experiments involving Holstein-Friesian cattle challenged with either bovine or ovine strains of Mycobacterium avium subsp. paratuberculosis (Map) have been conducted over a period of 54 and 35 months, respectively. Blood samples for the IFN-gamma test and the absorbed ELISA and faecal samples for bacteriological culture were taken pre-challenge and monthly post-challenge. Cell-mediated (CMI) responses were substantially higher for the bovine Map strain during the 42-month period following dosing but then declined in the remaining 12 months. However, for the ovine Map challenge and control groups, CMI responses were not significantly different from each other. None of the cattle developed clinical disease and only one of the cattle in the bovine Map gut mucosal tissue challenged group was a persistent faecal shedder and also an ELISA antibody responder which developed after shedding commenced. Culture of tissues, following necropsy at the completion of the experiments, showed no evidence of infection in any of the challenged cattle and sheep for either the bovine or ovine Map strain in contrast to positive cultures for challenged goats in the same experiments. The tissues from the control cattle, sheep and goats were culture negative. The cattle were less susceptible to the bovine and ovine Map strains than goats and sheep with the goats being the least naturally resistant.

Hendra and Nipah viruses are newly emerged, zoonotic viruses and their genomes have nucleotide and predicted amino acid homologies placing them in the family Paramyxoviridae. Currently these viruses are classified in the new genus Henipavirus, within the subfamily Paramyxovirinae, family Paramyxoviridae. The genes encoding HeV and NiV nucleocapsid proteins were cloned into the yeast Saccharomyces cerevisiae expression vector pFGG3 under control of GAL7 promoter. A high level of expression of these proteins (18-20 mg l(-1) of yeast culture) was obtained. Mass spectrometric analysis confirmed the primary structure of both proteins with 92% sequence coverage obtained using MS/MS analysis. Electron microscopy demonstrated the assembly of typical herring-bone structures of purified recombinant nucleocapsid proteins, characteristic for other paramyxoviruses. The nucleocapsid proteins revealed stability in yeast and can be easily purified by cesium chloride gradient ultracentrifugation. HeV nucleocapsid protein was detected by sera derived from fruit bats, humans, horses infected with HeV, and NiV nucleocapsid protein was immunodetected with sera from, fruit bats, humans and pigs. The development of an efficient and cost-effective system for generation of henipavirus nucleocapsid proteins might help to improve reagents for diagnosis of viruses.

Aims: To compare amidation and acylation of lactoferrin (LF) from bovine milk, as a means of enhancing its antimicrobial and antiviral properties., Methods and Results: LF was chemically modified by amidation with a 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide (EDC) in the presence of ammonium ions or by acylation with either succinic or acetic anhydride. In the test systems used, amidation substantially enhanced the activity of LF against Pseudomonas fluorescens in comparison with native LF. However, increasing the net negative charge of LF by acylation had no effect on the activity of LF against P. fluorescens, and abrogated the antimicrobial activity of LF against Bacillus subtilis and Saccharomyces cerevisiae. Increasing the net negative charges of LF by acylation eliminated its antimicrobial and antiviral effects against poliovirus and feline calicivirus (nonenveloped viruses)., Conclusions: The addition of positive charges to LF via amidation enhanced antimicrobial properties in contrast to increasing the negative charges by acylation, which abolished both the antimicrobial and antiviral properties of LF., Significance and Impact of the Study: The effects of charge alteration of LF determined in this study provides a basis for further development of LF formulations with enhanced antimicrobial effectiveness for use in food process hygiene, veterinary and health-care applications.

Photosystem I and Photosystem II activities, as well as polypeptide content of chlorophyll (Chl)-protein complexes were analyzed in mesophyll (M) and bundle sheath (BS) chloroplasts of maize (Zea mays L.) growing under moderate and very low irradiance. This paper discusses the application of two techniques: mechanical and enzymatic, for separation of M and BS chloroplasts. The enzymatic isolation method resulted in depletion of polypeptides of oxygen evolving complex (OEC) and alphaCF1 subunit of coupling factor; D1 and D2 polypeptides of PSII were reduced by 50%, whereas light harvesting complex of photosystem II (LHCII) proteins were still detectable. Loss of PSII polypeptides correlated with the decreasing of Chl fluorescence measured at room temperature. Using mechanical isolation of chloroplasts from BS cells, all tested polypeptides could be detected. We found a total lack of O2 evolution in BS chloroplasts, but dichlorophenolindophenol (DCPIP) was photoreduced. PSI activity of chloroplasts isolated from 14- and 28-day-old plants was similar in BS chloroplasts in moderate light (ML), but in low light (LL) it was reduced by about 20%. PSI and PSII activities in M chloroplasts of plants growing in ML decreased with aging of plants. In older LL-grown plants, activities of both photosystems were higher than those observed in chloroplasts from ML-grown plants. We suggest that in BS chloroplasts of maize, PSII complex is assembled typically for the agranal membranes (containing mainly stroma thylakoids) and is able to perform very limited electron transport activity. This in turn suggests the role of PSII for poising the redox state of PSI.

Two longitudinal experiments involving Angora goats challenged with either bovine or ovine strains of Mycobacterium avium subspecies paratuberculosis (Map) have been conducted over a period of 54 and 35 months, respectively. Blood samples for the interferon-gamma (IFN-gamma) test and the absorbed ELISA and faecal samples for bacteriological culture were taken pre-challenge and monthly post-challenge. Persistent shedding, IFN-gamma production, seroconversion and clinical disease occurred earlier with the bovine Map gut mucosal tissue challenge inoculum than with cultured bacteria. The IFN-gamma responses of the gut mucosal tissue and bacterial challenge groups were substantially and consistently higher than those of the control group. The in vivo and cultured cattle strains were much more pathogenic for goats than the sheep strains with persistent faecal shedding, seroconversion and clinical disease occurring in the majority of bovine Map challenged goats. With the ovine Map, 3 goats developed persistent antibody responses but only one of these goats developed persistent faecal shedding and clinical disease. However, there was no significant difference between the IFN-gamma responses of the tissue challenged, bacterial challenged and control groups. Compared with sheep, the ELISA appeared to have higher sensitivity and the IFN-gamma test lower specificity.

To assess the rabbit as a model for the study of Johne's disease pathogenesis, a breeding group of adult and juvenile New Zealand white rabbits were orally challenged with three doses of the Mycobacterium avium subspecies paratuberculosis wildtype bovine strain, CLIJ623, on three occasions. Faecal culture, post-mortem tissue bacteriological culture and histopathology were used to monitor the disease progression in the rabbits for more than 2 years. Of 4 adult and 16 juvenile orally dosed rabbits M. paratuberculosis organisms were recovered bacteriologically from two and three animals, respectively, using the BACTECtrade mark radiometric culture system. Tissue sites from which the bacteria were recovered included the mesenteric lymph nodes, ileocaecal valve, vermiform appendix, caecum, proximal colon and jejunum. Body weight loss, reduced abdominal fat and mild lesions were observed at necropsy in four infected rabbits. Diarrhoea and persistent faecal shedding of bacteria were not observed. Faecal culture did not yield any cultivable mycobacterial organisms on solid media.

Peptide mimotopes of plant-associated toxins offer the potential for improving analytical and diagnostic methodologies as well as providing candidates for potential protective vaccines against plant poisoning diseases. Monoclonal antibody (mAb) C3C11, which recognizes the antimicrotubule phomopsin mycotoxins, was used to isolate peptide mimics of phomopsin A from a random 15-mer phage display peptide library. A total of 46 clones were isolated that showed specific reactivity with the mAb. Amino acid sequence analysis revealed four different types of mimotope sequences, all of which contained a common motif V-A-L/V-C. Of the 46 clones isolated, 44 contained the motif V-A-L-C while 2 contained the V-A-V-C motif. All four types of phage clones inhibited the reactivity of the mAb with phomopsin A in a competition ELISA. The clone with the mimotope sequence CTVALCNMYFGAKLD demonstrated the strongest binding. It was further shown that synthetic peptides containing these mimotope amino acid sequences were able to inhibit the mAb-phomopsin A interaction, indicating that the peptide mimotopes were responsible for the specific binding, independent of the phage framework. The results also suggest that the mimotope peptides bind to mAb C3C11 at the same site as phomopsin A. The application of recombinant phage particles carrying phomopsin mimotopes in immunoassay was evaluated and the results demonstrated approximately 100-fold increase in sensitivity in comparison with a conventional immunoassay using a chemically linked phomopsin-horseradish peroxidase conjugate.

Two longitudinal experiments involving Merino sheep challenged with either bovine or ovine strains of Mycobacterium avium subsp. paratuberculosis (Map) have been conducted over a period of 54 and 35 months, respectively. Blood samples for the interferon-gamma test, the absorbed ELISA and faecal samples for bacteriological culture were taken pre-challenge and monthly post-challenge. Infections were induced with either a bovine or ovine strain of Map in separate experiments with infections being more easily established, in terms of faecal bacterial shedding and clinical disease when the challenge inoculum was prepared from gut mucosal tissue than cultured bacteria. The patterns of response for shedding and clinical disease were similar. Cell-mediated immune responses were proportionally elevated by at least an order of magnitude in all sheep dosed with either a bovine or ovine strain of Map. Conversely, antibody responses were only elevated in a relatively small proportion of infected sheep. Neither of the clinically affected tissue challenged sheep developed an antibody response despite the presence of persistent shedding and the development and decline in cell-mediated immunity. The results indicated that for sheep the interferon-gamma test may be useful for determining if a flock has been exposed to ovine Johne's disease.

Mycobacterium avium subsp. paratuberculosis has long been recognized as the causative agent of Johne's disease, a chronic inflammatory intestinal disease of sheep, cattle and other ruminants. Mycobacterial cells are extremely hardy, and proteomic analyses require the use of harsh conditions to effect their disruption. We compared the effectiveness of bead beating and sonication as cell lysis methods for the extraction of the proteomes of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis. Broad and narrow range two-dimensional gel electrophoresis was used to compare the numbers of silver stained protein spots that were observed in mycobacterial lysates. Despite differences in the yield of total protein from either species, and at different ages, the two methods appeared to give similar representations of the mycobacterial proteomes analyzed. Bead beating therefore represents a rapid and effective method of extracting the proteomes of mycobacterial species without the risks associated with an open tube sonication procedure.

Hendra virus (HENV) and Nipah virus (NIPV) are classified in the new genus Henipavirus, within the subfamily Paramyxovirinae, family Paramyxoviridae. The genetic and biological characteristics that differentiate henipaviruses from other members of the subfamily are summarized. Although they do not display neuraminidase and hemagglutination activities and in that regard resemble viruses in the genus Morbillivirus, several recent observations highlight similarities between henipaviruses and respiroviruses (genus Respirovirus) in structure and replication strategy. First, three-dimensional modeling studies suggest that the external globular head domain of the HENV G protein resembles that of respiroviruses rather than morbilliviruses. Second, the pattern of transcriptional attenuation in HENV-infected cells resembles that observed with Sendai virus, a respirovirus, and differs from that found in cells infected with measles virus, a morbillivirus. Henipaviruses have a broad host range in vitro and in vivo, indicating wide distribution of cellular receptor molecules. The extensive host range has been confirmed in a quantitative in vitro cell-fusion assay using recombinant vaccinia viruses expressing the attachment and fusion proteins of HENV and NIPV. Cell lines of diverse origin and which are permissive in the in vitro cell fusion assay have been identified and the pattern of relative susceptibilities is the same for both HENV and NIPV, implying that both viruses use the same cell receptor. Protease treatment of permissive cells destroys their ability to fuse with cells expressing viral envelope glycoproteins. Virus overlay protein binding assay (VOPBA) and radio-immune precipitation assays confirm that both HENV and NIPV bind to membrane proteins in the 35-50 kD range. Treatment of cell membrane proteins with N-glycosidase eliminates HeV binding activity in VOPBA whereas treatment with neuraminidase has no effect on binding. Thus preliminary evidence suggests that NIPV and HENV bind to the same glycoprotein receptor via a non-sialic acid-dependant mechanism.

Hendra (HeV) and Nipah (NiV) viruses are newly emerged, zoonotic viruses and their genomes have nucleotide and predicted amino acid homologies placing them in the subfamily Paramyxoviridae. The polymerase-associated phosphoproteins (P proteins) of paramyxoviruses have been shown, by direct and indirect methods, to be highly phosphorylated. In this study, a comprehensive comparison of in vivo phosphorylation of HeV and NiV P proteins, derived from virus particles, was achieved by a direct approach using electrospray ionization ion trap mass spectrometry (ESI-IT-MS). Phosphorylation sites for the P proteins were determined at Ser-224 and Thr-239 in HeV and at Ser-240 and Ser-472 in NiV. These phosphorylation patterns do not appear to be consistent with those reported for other paramyxoviruses. Protein V, a product of a frame shift in the P protein gene, was identified by specific antibodies in HeV preparations but not in NiV. HeV V protein was found to contain phosphoserine but not phosphothreonine. In addition, P proteins from both viruses were found to be modified by N-terminal acetylation.

The recent emergence of novel viruses requires reliable methodology for their identification and confirmation both on a cellular and molecular level. Mass spectrometry offers a suitable approach for the identification and characterisation of viral proteins and its application is demonstrated in this study. Menangle virus is a previously unclassified member of the family Paramyxoviridae isolated in Australia in 1997. Menangle virus caused disease in pregnant pigs, and like the other newly emergent Hendra, Nipah and Tioman viruses, appears to be a virus of fruit bats (flying foxes) in the genus Pteropus. The 61 kDa gel-purified protein isolated from cell-associated Menangle virus ribonucleoprotein (RNP) was identified as the nucleocapsid protein (NP) by peptide mapping, mass spectrometry and amino acid sequencing. Over 69% of the amino acid sequence was obtained and found to be identical with that derived from gene analysis (Virology, 283 (2001), 358). The first residue of the mature NP was found to be serine (second residue in the gene derived amino acid sequence). The NP was found to be acetylated at the N-terminus (at Ser-2) and appears to be not modified by phosphorylation.

A candidate for a vaccine against infectious bovine keratoconjunctivitis (IBK) has been cloned and characterized from Moraxella bovis. The plb gene encodes a protein of 616 amino acids (molecular mass of ~65.8 kDa) that expresses phospholipase B activity. Amino acid sequence analysis revealed that PLB is a new member of the GDSL (Gly-Asp-Ser-Leu) family of lipolytic enzymes.

An outbreak of acute respiratory disease in Hendra, a suburb of Brisbane, Australia, in September 1994 resulted in the deaths of 14 racing horses and a horse trainer. The causative agent was a new member of the family Paramyxoviridae. The virus was originally called Equine morbillivirus but was renamed Hendra virus (HeV) when molecular characterization highlighted differences between it and members of the genus Morbillivirus. Less than 5 years later, the closely related Nipah virus (NiV) emerged in Malaysia, spread rapidly through the pig population, and caused the deaths of over 100 people. We report the characterization of the HeV L gene and protein, the genome termini, and gene boundary sequences, thus completing the HeV genome sequence. In the highly conserved region of the L protein, the HeV sequence GDNE differs from the GDNQ found in almost all other nonsegmented negative-strand (NNS) RNA viruses. HeV has an absolutely conserved intergenic trinucleotide sequence, 3'-GAA-5', and highly conserved transcription initiation and termination sequences similar to those of respiroviruses and morbilliviruses. The large genome size (18,234 nucleotides), the unique complementary genome terminal sequences of HeV, and the limited homology with other members of the Paramyxoviridae suggest that HeV, together with NiV, should be classified in a new genus in this family. The large genome of HeV also fills a gap in the spectrum of genome sizes observed with NNS RNA virus genomes. As such, it provides a further piece in the puzzle of NNS RNA virus evolution.

Hendra virus (HeV) is an unclassified member of the Paramyxoviridae family that causes systemic infections in humans, horses, cats, guinea pigs and flying foxes. The fusion protein (F(0)) of members of the Paramyxoviridae family that cause systemic infections in vivo contains a basic amino acid-rich region at which the protein is activated by cleavage into two subunits (F(1) and F(2)). HeV F(0) lacks such a domain. We have determined the cleavage site in HeV F(0) by sequencing the amino terminus of the F(1) subunit and in view of the potential effect of glycosylation on the cleavage process have ascertained the sites at which F(0) is glycosylated. The results indicate that unlike other members of the family that replicate in cultured cells and cause systemic infections in vivo, cleavage of HeV F(0) occurs at a single lysine (reside 109) in the sequence Asp-Val-Lys- downward arrow-Leu. Although HeV genotypically resembles members of the Respirovirus and Rubulavirus genera in having potential N-linked glycosylation sites in both the F(1) and F(2) subunits, we show that phenotypically HeV may more closely resemble members of the Morbillivirus genus that contain N-linked glycans only in the F(2) subunit.

Type 4 fimbriae have been identified on the cell surface of Actinobacillus pleuropneumoniae by electron microscopy and N-terminal sequencing analysis. A. pleuropneumoniae type 4 fimbrial subunit protein, purified from cell cultures and from outer membrane preparations, reacted with polyclonal antibody raised against type 4 fimbriae of Moraxella bovis on Western blots. N-terminal sequence analysis of the purified 17 kDa type 4 fimbrial subunit protein, named ApfA, revealed the first 12 amino acids to be identical to those of other type 4 fimbrial subunit proteins.

A concentrated bacterial culture supernatant from the hemolytic Moraxella bovis strain UQV 148NF was used to immunize mice and generate monoclonal antibodies (MAbs). One, MAb G3/D7, neutralized the hemolytic activity of M. bovis and recognized a 94-kDa protein by Western blot analysis in hemolytic M. bovis strains representing each of the different fimbrial serogroups. Exposure of corneal epithelial cells to M. bovis concentrated culture supernatants demonstrated a role for an exotoxin in the pathogenesis of infectious bovine keratoconjunctivitis, while neutralization of hemolytic and cytotoxic activities by MAb G3/D7 implies that these activities are related or have common epitopes. The action of M. bovis hemolysin was further characterized in sheep erythrocyte preparations with a binding step and Ca(2+) required for lysis to proceed, similar to the RTX family of bacterial exotoxins. Neutralization of lytic activity in vitro is evidence for the presence of M. bovis antigens, which may be capable of protecting cattle from the development of infectious bovine keratoconjunctivitis.

Interferon-gamma (IFN-gamma) possesses potent immunostimulatory properties, and it has recently been shown to have potential therapeutic properties. Recombinant protein technology is frequently used for commercial production of therapeutics, such as IFN. Biologically active recombinant chicken IFN-gamma (rChIFN-gamma) constructs bearing an N-terminal poly-His tag were expressed in Escherichia coli. Preparations of rChIFN-gamma contained varying ratios of a full-length and a truncated protein species (18 and 16 kDa, respectively). Amino acid sequence analysis of the full-length protein corroborated the sequence previously predicted from the cDNA sequence. Full-length rChIFN-gamma contains two cysteine residues at the C-terminus, and these were labeled by reduction and subsequent specific alkylation with fluorescent tag (5-I-AEDANS) to distinguish between full-length and C-terminally truncated forms of rChIFN-gamma. Comparative peptide mapping, amino acid sequencing, and mass spectrometry revealed that the 16 kDa protein was truncated at Lys133. It was also observed that the 18 kDa rChIFN-gamma protein was infrequently contaminated with small quantities of protein truncated at Arg141. A truncated recombinant construct (His1-Lys133) was also expressed in E. coli and had biologic activity comparable with that of the full-length construct. The 3-D structure of rChIFN-gamma was deduced by comparative modeling with bovine and human IFN-gamma crystallographic structures. Analysis of sequences and comparison of structures have revealed that the 3-D structure of rChIFN-gamma is similar to those of bovine and human molecules despite an overall amino acid identity of only 32%.

In 1994, a new member of the family Paramyxoviridae isolated from fatal cases of respiratory disease in horses and humans was shown to be distantly related to morbilliviruses and provisionally called equine morbillivirus (K. Murray et al., Science 268:94-97, 1995). To facilitate characterization and classification, the virus was purified, viral proteins were identified, and the P/V/C gene was cloned and sequenced. The coding strategy of the gene is similar to that of Sendai and measles viruses, members of the Paramyxovirus and Morbillivirus genera, respectively, in the subfamily Paramyxovirinae. The P/V/C gene contains four open reading frames, three of which, P, C, and V, have Paramyxovirinae counterparts. The P and C proteins are larger and smaller, respectively, than are cognate proteins in members of the subfamily, and the V protein is made as a result of a single G insertion during transcription. The P/V/C gene has two unique features. (i) A fourth open reading frame is located between those of the C and V proteins and potentially encodes a small basic protein similar to those found in some members of the Rhabdoviridae and Filoviridae families. (ii) There is also a long untranslated 3' sequence, a feature common in Filoviridae members. Sequence comparisons confirm that although the virus is a member of the Paramyxovirinae subfamily, it displays only low levels of homology with paramyxoviruses and morbilliviruses and negligible homologies with rubulaviruses.

The presence of fimbriae on Pasteurella multocida has been reported, but there have been no prior studies aimed at conclusively characterizing these structures. We now report on the identification and characterization of type 4 fimbriae on serogroup A, B, and D strains of P. multocida. Under microaerophilic conditions P. multocida showed an increased expression of the fimbriae, which were observed to form bundles. Fimbriae purified by high-performance reverse-phase liquid chromatography constituted a single 18-kDa subunit, the first 21 amino acids of which shared very high similarity with the N-terminal amino acid sequence of other type 4 fimbrial subunits. Antiserum against the P. multocida 18-kDa protein immunostained the type 4 fimbrial subunit of Moraxella bovis and Dichelobacter nodosus. Based on these observations we conclude that P. multocida possesses type 4 fimbriae and have designated the P. multocida fimbrial subunit PtfA.

A brief overview of the family of superoxide dismutase (SOD) enzymes and their biomedical significance is presented. Methodology for the purification and electrophoretic analysis of superoxide dismutases is reviewed and discussed, with emphasis on the specific problems raised by the separation of individual superoxide dismutase isoenzymes. Purification methods and their performance, as reported in the literature are summarised in table form. Generally used methods for measuring SOD activity in vitro and SOD visualisation after electrophoresis are outlined, particularly those relevant to the monitoring of progress of SOD purification.

For accreditation of CSIRO specific pathogen-free flocks, chicken sera are routinely tested for antibody to chicken anaemia virus (CAV) using a commercially available ELISA kit. On some occasions recently, up to 18.2% positive reactions have been found in individual isolators, with an overall reaction rate of 2.7%. On subsequent bleeding the majority of the suspected birds have given negative reactions, indicating that the earlier reactions were false-positives. The protein composition of the sera that appeared CAV-negative or -positive in the ELISA were compared using various Chromatographic techniques. Separation of serum proteins by high resolution size exclusion chromatography revealed that in the false-positive sera the antigen-binding activity is associated with a protein fraction which was different from that found in the true-positive sera.

Protection conferred by a cell-free preparation from a haemolytic Moraxella bovis isolate, UQV 148NF, was compared to an equivalent fraction from a non-haemolytic M. bovis isolate, Gordon 26L3, and to a recombinant DNA-derived pili vaccine. Three groups of ten calves were vaccinated twice with one of the three preparations and, together with ten non-vaccinated calves, challenged with virulent M. bovis isolate Dal 2d. Compared to the control group, significant protection was observed in the group receiving the pili vaccine and the group receiving the preparation from haemolytic isolate, UQV 148NF.

Homogenates of sporulated oocysts of E. tenella have detectable proteolytic activity which is completely inhibited by phenylmethylsulfonyl fluoride and L-trans-epoxysuccinyl-leucyl-amido-(4-guanidino)-butane, indicating the presence of both serine and cysteine-type proteases in sporulated oocysts. A serine-type protease has been purified from the homogenate using immobilised-bacitracin affinity chromatography. The monomeric enzyme had an apparent M(r) of 20,000 and a pI of 8.6. The maximum proteolytic activity with azocasein and gelatin was observed at pH 8.0. Antibodies raised in rabbits and chickens against purified protease recognised this protein on blots of sporozoite homogenates.

A group of glycoproteins, which strongly bind peanut agglutinin (PNA) was found in Eimeria tenella. Two major antigenic glycoproteins, Et110gp and Et35gp, were identified in sporulated oocysts and sporozoites. Molecular characterisation of carbohydrate moieties (lectin binding, enzymic hydrolysis and monosaccharide composition) revealed that both glycoproteins are rich in galactose and N-acetylgalactosamine, and appear to be sialylated. Both glycoproteins were susceptible to treatment with neuraminidase followed by O-glycosidase, suggesting that the oligosaccharide chains are attached to the protein by an O-glycosidic linkage to serine and/or threonine. Purified Et35gp contained a large number of serine (14) and threonine (33) residues, and was rich in glycine. This protein aggregated after repetitive lyophilisation and migrated on SDS-PAGE gels as an 85,000 protein. Sera against purified Et35gp raised in chickens and rabbits, and anti-E. tenella immune chicken serum recognised both antigens on blots and on the surface of sporozoites. Chickens immunised with purified Et35gp were not protected against coccidial infection.

Unsporulated oocysts of Eimeria tenella contain large quantities of carbohydrates, namely amylopectin, mannitol and glucose. Analysis of carbohydrate content of sporulating oocysts revealed that mannitol content increased markedly during early stages of sporogony (first 4-6h) but slowly diminished during the next 40h of sporulation. Accumulation of mannitol was accompanied by a rapid decrease in amylopectin and free glucose, suggesting that mannitol might be synthesized from glucose released from amylopectin. Mannitol was also detected in sporozoite and merozoite extracts. All four mannitol cycle enzymes were detected in oocysts. Sporozoites excysted in vitro had lower activities of all four enzymes. Mannitol-1-phosphatase and mannitol dehydrogenase activity was also detected in merozoites obtained from the second stage schizonts. Sporozoites incubated with 14C-glucose accumulated radioactively labelled precursor continuously for over 12h and some of the 14C-glucose was converted into 14C-mannitol. These results indicate that mannitol plays an important role in the metabolism and development of the intracellular stages of the parasite.

A new method for separation of three forms of superoxide dismutase (SOD) using immobilised metal affinity chromatography (IMAC) is reported. Fe-, Mn- and Cu/Zn-SODs were eluted sequentially from Cu(2+)-IMAC column with an increasing gradient of a counter ion (NH+4) run in combination with an increasing pH gradient (6.8-7.8). The combined gradient elution method resulted in separation of SODs with high resolution, the three proteins being eluted in electrophoretically homogeneous forms. Similar preparation could not be achieved by either increasing gradient of a counter ion or decreasing pH gradients used separately. The described methodology has been successfully applied for separation of three SODs from a protozoan parasite, indicating that this combined gradient elution system for IMAC offers new possibilities for the high-resolution separation of proteins exhibiting only minor differences in their amino acid composition and structure.

Cell-mediated immunity is thought to be important in the resistance of chickens to infection by coccidia, and it has been demonstrated that sporozoites of Eimeria tenella are very sensitive to superoxide ions. Therefore an investigation into the cellular responses in naive specific pathogen-free and hyperimmune birds was carried out with particular attention to their ability to produce reactive derivatives of oxygen. Leucocytes were isolated from the blood, spleen and caecal mucosa of chickens infected with E. tenella and assessed for their ability to release H2O2. Leucocytes obtained from the blood and spleen of hyperimmune birds 1 day after challenge showed an elevated ability to produce reactive oxygen intermediates. In contrast, the ability of leucocytes from naive chickens to produce these molecules was transiently depressed after challenge. Prior to challenge, mucosal leucocytes from immune chickens were also able to release heightened levels of H2O2 when compared with cells from naive chickens.

Unsporulated oocysts of Eimeria tenella have high superoxide dismutase (SOD: superoxide:superoxide oxidoreductase, EC 1.15.1.1.) activity and contain several electrophoretically distinct forms of the enzyme, including two forms of Cu/Zn-containing SOD, two forms of Fe-SOD and two forms of Mn-SOD. SOD activity remains high during 12 h of sporulation but diminishes slowly during prolonged sporulation. Oocysts sporulated for 48 h have low levels of superoxide dismutase and contain only one form of the enzyme (Mn-SOD), which was also found in sporozoites. In vitro, sporozoites are oxidant-sensitive and die within minutes of superoxide radical (O2-) generation but SOD/catalase and mannitol protect sporozoites against oxidative damage. These data suggest that E. tenella sporulated oocysts and sporozoites lack soluble cytoplasmic SOD and that this deficiency may contribute to the oxidant sensitivity of the parasite.

1. Copper, zinc superoxide dismutase (Cu,Zn SOD) has been purified to homogeneity from chicken erythrocytes by anion-exchange, immobilized metal affinity and size exclusion chromatography. 2. Molecular properties (amino acid composition, molecular mass, subunit composition and spec. act.) of the chicken enzyme are similar to those of a bovine erythrocyte Cu,Zn SOD. 3. The chicken and bovine enzymes are immunologically similar since antisera raised against each enzyme are cross-reactive.

1. Both galactolipase treatment of tomato chloroplasts and the cold and dark storage of leaves induce a large degradation of chloroplast monogalactosyl diacylglycerol and digalactosyl diacylglycerol as well as an accumulatwon of free fatty acids accompanied by the inhibition of Hill reaction activity with water as electron donor. All these changes are reversed upon illumination of the leaves. 2. Inhibition of diphenylcarbazide (DPC) leads to dichlorophenolindophenol (DCIP) activity by free fatty acids released following galactolipase treatment of chloroplasts isolated from either fresh or cold and dark-stored and illuminated leaves is almost completely reversed by either bovine serum albumin or Mn2+, while that in chloroplasts from the cold and dark-stored leaves is reversed by bovine serum albumin and Mn2+ only up to about 60 and 25%, respectively. 3. Fatty acids released during the treatment of chloroplasts with galactolipase affect the electron transport mainly in the same site as exogenous unsaturated fatty acids do, while those released due to endogenous galactolipase activity appear to affect also in the region damaged by either Tris washing of chloroplasts or the cold and dark treatment of leaves. 4. The loss of manganese from chloroplasts (Kaniuga, Z., Zabek, J. and Sochanowicz, B. (1978) Planta 144, 49-56) seems to be the main reason of cold and dark-induced inactivation of Hill reaction activity in chloroplasts of chilling-sensitive plants, while both the degradation of galactolipids and the accumulation of fatty acids are of secondary importance.

1. The role of tocopherols in tomato chloroplasts from fresh, cold and dark-stored as well as stored and illuminated leaves was studied. 2. The cold and dark storage of leaves results in a loss of chloroplast alpha- and gamma-tocopherols of about 30-40% accompanied by an increase in chloroplast delta-tocopherol of about 40%. On illumination of stored leaves, an elevation of alpha- and gamma-tocopherol level to about 110 and 95% of the control, respectively, occurs, whilst delta-tocopherol content is not affected. 3. Experiments performed with 2,2-diphenyl-1-picrylhydrazyl-treated chloroplasts show that only about 70% of total alpha-tocopherol is functionally active in the electron transport of Photosystem II between the diphenylcarbazide (DPC) donation site and the inhibition site of DBMIB. 4. A small amount of alpha-tocopherol quinone (about 10% of alpha-tocopherol content) is found in chloroplasts from fresh, fresh and illuminated as well as cold and dark-stored tomato leaves, whereas the illumination of the latter increases the chloroplast alpha-tocopherol quinone content 3-fold. Moreover, following the illumination of chloroplasts from cold and dark-stored as well as stored and illuminated leaves, the oxidation of exogenous alpha-tocopherol to alpha-tocopherol quinone is 2-fold faster then in chloroplasts from fresh leaves. 5. The primary product ('alpha-tocopheroxide') formed during the alpha-tocopherol oxidation by illuminated chloroplasts was identified as 8a-hydroxy-alpha-tocopheron. 6. Exogenous alpha-tocopherol inhibits the lipid photoperoxidation by about 40-50% in chloroplasts from all three kinds of tomato leaf. 7. The results seem to suggest that chloroplast alpha-tocopherol is involved in both electron transport of PS II and antioxidizing system of chloroplasts.

Both total and CN-insensitive superoxide dismutase (SOD) activities in relation to the level of manganese were studied in chloroplasts and digitonin subchloroplast particles obtained from fresh, cold and dark-stored, and illuminated leaves of Licopersicon esculentum, Mill. Following cold and dark treatment of detached leaves and intact plants, both total SOD activity and Mn content were greatly diminished while illumination of the leaves (2 h, 8,000 lx, 25° C) resulted in a large increase of Mn content and a partial restoration, of SOD activity. When growing plants were kept for 3-4 days in the dark, either at 0° C or 25° C, a decrease in chloroplast SOD activity, Mn content, and Hill reaction was also observed. However, with a prolonged dark treatment either at 0° C or at 25° C, there was a slight increase in chloroplast Mn content accompanied by an enhancement of both SOD and Hill reaction activities, suggesting that even in the dark some translocation of Mn from cytosol into chloroplasts may occur. Hypotonically treated chloroplasts and digitonin subchloroplast particles contained almost exclusively CN-insensitive, SOD activity, while Tris-washed chloroplast preparations were completely deprived of SOD activity. All these data seem to indicate that a chloroplast loosely-bound Mn pool is involved in CN-insensitive SOD activity.

The inclusion of nitrate or nitrite in cultures of Rhodobacter spaeroides subsp. denitrificans grown heterotrophically in light depressed the formation of bacteriochlorophyll a. The pigment biosynthesis was inhibited at the stage of the reduction of chlorophyllide (chlorin) to bacteriochlorophyllide (tetrahydroporphyrin) since 3-hydroxyethylchlorophyllide a accumulated in the culture medium. The addition of exogenous 5-aminolevulinic acid to these cultures resulted in a complete restoration of bacteriochlorophyll synthesis accompanied by the accumulation of 3-vinylbacteriopheophorbide. This indicates that under these conditions bacteriochlorophyll was formed via an alternative route, in which the reduction of chlorins to tetrahydroporphyrins precedes modifications of the C-3 side chain. The multiple forms of 5-aminolevulinic acid synthase were purified from cells grown with and without nitrate. Antibodies against these proteins were raised in rabbits and used in enzyme-linked immunosorbent assays for various forms of 5-aminolevulinic acid synthase. In denitrifying cells, the amount and activity of fraction I of the enzyme was reduced by approximately 40 and 30%, respectively. Partly active enzymes from both types of cells were activated by cystine trisulfide.