Occurrence of blood parasites in seabirds admitted for rehabilitation in the Western Cape, South Africa, 2001-2013.

Blood parasites are generally uncommon in seabirds, and knowledge on their epidemiology is further limited by the fact that they often inhabit remote locations that are logistically difficult or expensive to study. We present a long term data set of blood smear examinations of 1909 seabirds belonging to 27 species that were admitted to a rehabilitation centre in Cape Town (Western Cape, South Africa) between 2001 and 2013. Blood parasites were detected in 59% of species (16/27) and 29% of individuals examined (551/1909). The following blood parasites were recorded: Babesia ugwidiensis, Babesia peircei, Babesia sp., Plasmodium sp., Leucocytozoon ugwidi, Hepatozoon albatrossi, Haemoproteus skuae and Spirochaetales. Several of the records are novel host-parasite associations, demonstrating the potential of rehabilitation centres for parasite and disease surveillance, particularly for species infrequently sampled from which no host-specific parasites have been described.

Babesia ugwidiensis, a new species of avian piroplasm from Phalacrocoracidae in South Africa.

A new species of haematozoa, Babesia ugwidiensis sp. nov. from a cormorant is described. This is the first species of piroplasm to be recorded from the Phalacrocoracidae and the relationship of this parasite to other Babesia spp. from marine hosts is discussed.

Inhibition of CD40-mediated endothelial cell activation with antisense oligonucleotides.

BACKGROUND: CD40-CD154 interactions play a pivotal role in the amplification of immune responses and, as such, represent an attractive target for immune intervention in a number of disease indications. We have previously shown that binding of human CD154 expressed on the Jurkat D1.1 cell line to porcine CD40 on pig aortic endothelial cells (PAECs) can lead to up-regulation of vascular cell adhesion molecule (VCAM)-1 and MHC class II. This activation can be completely inhibited by the addition of a monoclonal antibody (mAb) to human CD154. In this study, we explore an alternative approach to blocking this pathway with antisense oligonucleotides (ASOs). METHODS: Ten ASOs were generated on the basis of the porcine CD40 cDNA sequence. The ASOs that were found to reduce CD40 expression on PAECs were analyzed for their ability to reduce CD40-mediated PAEC activation. RESULTS: Four ASOs were found to significantly lower surface expression of porcine CD40 on PAECs 48 hr after transfection. Eight of the ASOs were seen to lead to mRNA cleavage products by ribonuclease protection assay. Of the four ASOs tested in the PAEC activation assay, one (ASO-9) showed a dramatic inhibition of PAEC activation (IC50 approximately 1 nM) results comparable to the use of a blocking mAb. Furthermore, we compared the effect of CD40 ASO on tumor necrosis factor alpha receptor signaling, in which we observed no effect, which confirmed ASO specificity. CONCLUSIONS: These results indicate that a CD40-dependent activation pathway can be inhibited with an ASO with high potency and specificity. ASO could be an attractive alternative therapy to the use of mAbs.

Uptake of metabolites by gonococci grown with lactate in a medium containing glucose: evidence for a surface location of the sialyltransferase.

Promotion of uptake of essential metabolites is a possible reason for the general stimulation of gonococcal metabolism which is caused by lactate (1 mM) in a defined medium containing glucose (5 mM). However, although uptake of(14)C adenine by gonococci [strain BS4(agar)] held for 4 or 7 min at 37 degrees C in Hanks balanced salt solution was increased for lactate treated gonococci compared with control organisms, uptake of(14)C glucose and(14)C proline under these conditions was unaffected. Hence, there is no evidence that lactate produces general stimulation of metabolite uptake. Unlike the other metabolites, cytidine 5'-monophospho-(14)CN-acetyl neuraminic acid (CMP-(14)CNANA), the substrate for sialylation of gonococcal lipopolysaccharide (LPS), was adsorbed in substantial quantities by gonococci held on ice for 6 min. Also, the increase in uptake of CMP-(14)CNANA at 37 degrees C over that adsorbed at 0 degrees C was much smaller (less than two-fold) than for the other compounds (4-30-fold). The substantial adsorption at 0 degrees C suggested a surface receptor for CMP-(14)CNANA. It is probably the sialyltransferase because a sialyltransferase deficient mutant, JB1, did not absorb CMP-(14)CNANA at 0 degrees C or take it up at 37 degrees C, in contrast to its parent strain, F62, which behaved similarly to strain BS4 (agar). This supports previous evidence for a surface location of the sialyltransferase. There was a small increase in adsorption of CMP-(14)CNANA in lactate treated gonococci indicating a slight increase in the surface enzyme. This could enhance LPS sialylation and hence affect pathogenicity.

Lactate causes changes in gonococci including increased lipopolysaccharide synthesis during short-term incubation in media containing glucose.

Gonococci (strain BS4(agar)), emerging from lag-phase during 1-1.5 h incubation in a medium containing glucose (28 mM) and either 5 microM or 50 microM sodium lactate, show enhanced capacity for their lipopolysaccharide (LPS) to be sialylated by cytidine 5'-monophospho-N-acetyl neuraminic acid. The sialyltransferase content of the lactate-treated gonococci was not greater than that of control organisms and showed no differences in LPS components. However, the total LPS content of the lactate-treated gonococci was 10-20% higher than that of control organisms, so lactate enhancement may be due to more sialyl receptors becoming available due to an overall stimulation of LPS synthesis. The protein and pentose contents of the lactate-treated gonococci were also higher than those of controls, indicating stimulation of protein synthesis and ribosome production. Electron microscopy showed hair-like external appendages on control but not on lactate-treated gonococci. The above growth conditions are unnatural. However, when concentrations of glucose and lactate were adjusted to values akin to those occurring in vivo (glucose 5 mM alone and with either 1 mM or 10 mM lactate), and gonococcal multiplication occurred during the short incubation period (1-1.5 h), lactate again induced greater contents of LPS, protein and pentose. A high content of LPS, which will contribute to pathogenicity, should be a constant feature of gonococci growing in human urogenital tissues, where lactate is ever present with glucose.

Lactate enhancement of sialylation of gonococcal lipopolysaccharide and of induction of serum resistance by CMP-NANA is not due to direct activation of the sialyltransferase: metabolic events are involved.

Lactate enhances lipopolysaccharide (LPS) sialylation and induction of serum resistance in gonococci by CMP-NANA. To investigate whether the enhancement is due to a direct effect on the sialyltransferase, an improved extraction of the enzyme and a reliable quantitative assay were devised. Gonococci (strain F62) were disrupted in a French pressure cell and the bacterial membranes were extracted for 1 h at 37 degrees C with a detergent, NONIDET (1% v/v). The assay involved sialylation of LPS by CMP-14CNANA and scintillation counting of the labelled LPS after fixing it on filter paper strips by trichloracetic acid (TCA) and washing away unincorporated CMP-14CNANA. It was rapid, reproducible and, although the enzyme preparations contained endogenous LPS, was dependent upon added LPS for maximum activity. At 37 degrees C the rate was constant for up to 5 min and proportional to the concentration of extract in the assay. A wide range of concentrations of lithium-L-lactate did not enhance the activity of the extracted sialyltransferase. At concentrations above 22 microM, it was inhibitory. Pre-incubation of gonococci with lactate enhanced subsequent LPS sialylation and induction of serum resistance by CMP-NANA. Hence, the process whereby lactate enhances the effect of CMP-NANA is separate from the action of CMP-NANA itself. Both processes were inhibited by a sublethal concentration of chloramphenicol, indicating that metabolic events are required. Evidently, the enhancement process does not involve a direct activation of the sialytransferase.

Functional characterization of a sialyltransferase-deficient mutant of Neisseria gonorrhoeae.

Previous studies indicate that sialylation of lipopolysaccharide (LPS) by host CMP-N-acetylneuraminic acid (CMP-NANA) catalyzed by bacterial sialyltransferase rendered gonococci resistant to killing by phagocytes, to entry into epithelial cell lines, to killing by immune serum and complement, and to absorption of complement component C3. These results have been confirmed by comparing a sialyltransferase-deficient mutant (strain JB1) with its parent (strain F62) in appropriate tests. In contrast to F62, JB1 was very susceptible to killing by human polymorphonuclear phagocytes in opsonophagocytosis tests and incubation with CMP-NANA did not decrease the level of killing. The inherent resistance of F62 in these tests was probably due to LPS sialylation by CMP-NANA and lactate present in the phagocytes. A JB1 variant expressing the invasion-associated Opa protein was as able to enter Chang human conjunctiva epithelial cells as an Opa-positive variant of F62, suggesting that the sialyltransferase is not required for Opa-mediated entry. After incubation with CMP-NANA, the number of F62 variant gonococci entering cells but not that of JB1 variant gonococci was drastically reduced. Both JB1 and F62 were killed by incubation with rabbit antibody to gonococcal major outer membrane protein, protein I, and human complement, but only F62 was rendered resistant to the killing by incubation with CMP-NANA. Finally, both JB1 and F62 absorbed similar amounts of complement component C3 and the binding was decreased by incubation with CMP-NANA only for the wild type, F62.

Lactic acid is the factor in blood cell extracts which enhances the ability of CMP-NANA to sialylate gonococcal lipopolysaccharide and induce serum resistance.

In previous work, a factor which enhances the ability of cytidine 5'-monophospho-N-acetyl neuraminic acid (CMP-NANA) to sialylate gonococcal lipopolysaccharide (LPS) was liberated at 4 degrees C in diffusates from high M(r) fractions of blood cell sonicates. The diffusates also contained CMP-NANA and converted serum susceptible gonococci to resistance. The enhancer has now been separated from CMP-NANA and material absorbing at 260 nm by HPLC on mu Bondapak-10 NH2. Resistance inducing activity was found only in fractions containing CMP-NANA and recovery was poor (about 25%). However, addition of enhancer fractions to CMP-NANA substantially increased its resistance inducing activity. Blood cell sonicates dialysed at 18-20 degrees C released enhancer in diffusates. These were ultrafiltered (nominal cut off 3000 Da) and fractionated on Biogel P2 which removed saccharides and most material absorbing at 260 nm. Over 90% of a fraction which was enhancer-active in nanogram quantities was identified by nuclear magnetic resonance (NMR) spectroscopy and gas chromatography/mass spectometry (GC/MS) as lactic acid. A fraction with similar properties was obtained from a different batch of diffusate by fractionation on Dowex 1. Authentic lithium L-lactate in nanogram quantities enhanced LPS sialyation by CMP-NANA and increased its serum resistance inducing activity. These results have important implications for gonococcal pathogenicity.

Sialylation of lipopolysaccharide by CMP-NANA in viable gonococci is enhanced by low Mr material released from blood cell extracts but not by some UDP sugars.

Serum resistance of gonococci in most patients is due to sialylation of a Gal beta 1-4GlcNAc group on a conserved 4.5 kDa lipopolysaccharide (LPS) component by host cytidine 5'-monophospho-N-acetyl neuraminic acid (CMP-NANA) catalysed by a gonococcal sialyl transferase. This sialylation is enhanced by a low M(r) factor(s) which, like CMP-NANA, is released in diffusates from high M(r) fractions obtained from sonicates dialysed at 4 degrees C. Also, as shown here, this factor(s) is released when the sonicates are dialysed at 18-20 degrees C. The enhancement of sialylation, first demonstrated using enzymes in gonococcal extracts, has been shown to occur in live gonococci and hence probably to have a role in pathogenicity. Gonococci, emerging from lag phase and incubated for 2 h with CMP-14CNANA fixed up to 90% more radiolabel than controls when the second factor(s) was present; their LPS separated by SDS-PAGE contained more radiolabel than control samples and label was not detected in any other component. Fractions with enhancing activity absorbed maximally at about 260 nm but a mixture of UDP-galactose (UDP-Gal), UDP-N-Acetyl galactosamine (UDP-GalNAc), UDP-glucose (UDP-Glc) and UDP-N-Acetyl glucosamine (UDP-GlcNAc) showed no significant enhancing activity. The enhancing action of the low M(r) fractions was unaffected by incubation with beta-galactosidase.

Identification by mass spectrometry of CMP-NANA in diffusible material released from high M(r) blood cell fractions that confers serum resistance on gonococci.

In previous work, a low M(r) component from human blood which converts serum-sensitive gonococci to resistance was shown to be indistinguishable from cytidine 5'-monophospho-N-acetyl neuraminic acid (CMP-NANA) by seven criteria. However, the presence of CMP-NANA was not proved by physicochemical methods. Purified, high M(r) fractions from human blood cells, which confer serum resistance on gonococci and enhance the transfer of sialyl groups from CMP-NANA to lipopolysaccharide (LPS) by a sialyltransferase in gonococcal extracts, were rechromatographed on DEAE Sepharose CL-6B. Both activities co-eluted from the column but on dialysis were found in the diffusate. After desalting the diffusate with Sephadex G10, the presence of CMP-NANA was proved by mass spectrometry. This confirmed previous work and is the first unequivocal demonstration of CMP-NANA in constituents of human blood cells.

The sialylation of gonococcal lipopolysaccharide by host factors: a major impact on pathogenicity.

The resistance of gonococci in most patients to complement mediated killing by human serum is due to sialylation of their lipopolysaccharide (LPS) which prevents bactericidal antibody from reacting with target sites. Two of the host factors responsible are: cytidine 5'-monophospho-N-acetyl neuraminic acid (CMP-NANA), a well-known sialylating agent, and another factor which enhances the transfer of sialyl groups from CMP-NANA to LPS catalysed by a gonococcal sialyltransferase. The bacterial determinant of resistance is a conserved LPS component of about 4.5 kDa which is sialylated at a terminal Gal beta 1-4GlcNAc site on its side chain. The sialylated LPS forms a surface coat which is stainable by ruthenium red and connected with previously described 'capsules'. These observations sparked off an explosion of research. Recent publications show that sialylation of LPS by CMP-NANA affects additional important aspects of gonococcal pathogenicity, notably interactions with antibodies and phagocytes, and rendering the gonococcal surface more 'host-like'. Also, the observations have prompted an examination of LPS from some other pathogens for the presence of sialyl groups with positive results for Neisseria meningitidis and Haemophilus influenzae.

A high Mr factor in human blood which confers serum resistance on gonococci: some properties and synergism with CMP-NANA.

A high relative molecular mass (M(r)) component which confers serum resistance on gonococci has been purified about 300-fold from a dialysed sonicate of human blood cells. Serum resistance conferred by the high M(r) factor (RIF), like that induced by cytidine-5' monophospho-N acetyl neuraminic acid (CMP-NANA), decreased when gonococci were incubated with neuraminidase. Also, the resistance-inducing activities of both high M(r) RIF and CMP-NANA were inhibited by CMP and inactivated at pH 4.0. These activities were not additive but synergistic. Neuraminidase decreased the activity of high M(r) RIF but not CMP-NANA. In tests with 14C CMP-NANA and gonococcal lipopolysaccharide, no sialyltransferase activity was detected, even in highly active samples of high M(r) RIF under conditions in which low activities of rat liver sialyltransferase were readily detected. Conversely, rat liver sialyltransferase was neither active in the RIF assay nor able to enhance the RIF activity of CMP-NANA. Nevertheless, high M(r) RIF greatly enhanced the sialyltransferase activity of a gonococcal extract; this enhancement suggests an explanation for the synergism between CMP-NANA and high M(r) RIF in inducing serum resistance in gonococci.

The serum resistance of gonococci in the majority of urethral exudates is due to sialylated lipopolysaccharide seen as a surface coat.

Examined before subculture, gonococci in 18 urethral exudates collected from different patients were serum-resistant. For 15 exudates, the resistance was drastically reduced by treatment with neuraminidase and by one subculture on laboratory media. It was restored by incubation with cytidine 5'-monophospho-N-acetyl neuraminic acid (CMP-NANA). Electron microscopic examination of gonococci in eight exudates showed a surface structure stained by Ruthenium red which disappeared in most samples when they were treated with neuraminidase. These results were identical with those of previous studies on in vitro grown gonococci which had shown that serum resistance is due to sialylation of a 4.5-kDa conserved component of gonococcal lipopolysaccharide (LPS) by host CMP-NANA, which masks the target site for bactericidal IgM and renders surface LPS stainable by Ruthenium red. The serum resistance of gonococci in the remaining three exudates was not reduced by neuraminidase nor by subculture. The mechanism of this stable resistance is unknown.

The surface structure seen on gonococci after treatment with CMP-NANA is due to sialylation of surface lipopolysaccharide previously described as a 'capsule'.

Phenotypic serum resistance of gonococci in urethral exudates is due to sialylation of lipopolysaccharide (LPS) by host cytidine 5'-monophospho-N-acetyl neuraminic acid (CMP-NANA). A surface structure was visible on gonococci [strain BS4 (agar)] that had been stained with ruthenium red after incubation with CMP-NANA. This structure was not visible after neuraminidase treatment, which released sialic acid but not LPS. The LPS profiles of strain BS4 (agar) had another in vivo-selected strain Gc40 (variant D1), were similar. A monoclonal antibody (mAb) which recognises epitope C on the LPS of 'capsulated' gonococci was shown by immunoblotting to react with several LPS components, including one of about 4.5 kDa which contains the probable site of sialylation by CMP-NANA. The reactions with the mAb were not affected by growing the strains with CMP-NANA nor by neuraminidase treatment of the sialylated LPS. The mAb also gold-labelled the surface of both strains before and after treatment with CMP-NANA. These data indicate that sialylation by CMP-NANA and staining with ruthenium red renders more visible the surface LPS which, sometimes in the past, has been seen as a 'capsule'.

Resistance to human serum of gonococci in urethral exudates is reduced by neuraminidase.

Gonococci examined directly from urethral exudates are resistant to killing by human serum, but most strains become susceptible on subculture. Previous work with gonococci grown in vitro indicates that resistance in vivo is due to sialylation of gonococcal lipopolysaccharide (LPS) by a host factor, cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NANA) or a related compound present in urogenital secretions and blood cells including phagocytes, which exude during inflammation. This sialylation inhibits the reaction between bactericidal IgM in serum and its target LPS sites. Here, we confirm the indication by using gonococci grown in vivo. Crucial to the above conclusions was the marked reduction of CMP-NANA-conferred serum resistance when gonococci were treated with neuraminidase to remove sialyl groups from their LPS. We now show that the serum resistance of gonococci in urethral exudates was reduced by treatment with neuraminidase from more than 95% (calculated in relation to controls incubated with heated serum) to 2-11% according to sample and incubation time. Subculture of the gonococci also reduced resistance to 9-11% but resistance was restored to more than 95% by incubation with CMP-NANA. This work is the culmination of an investigation that underlines the need to identify specific host factors and the virulence determinants they induce in vivo in future studies of pathogenicity.

In vitro and in vivo modification of Neisseria gonorrhoeae lipooligosaccharide epitope structure by sialylation.

After growth of gonococci in the presence of cytidine monophospho-N-acetyl-neuraminic acid (CMP-NANA), their 4.5-kD lipooligosaccharide (LOS) component was increased by approximately 400 daltons, whereas the LOS of strains lacking the 4.5-kD component were unaffected. Expression of mAb-defined epitopes on the 4.5-kD component was decreased on LOS of strains grown in CMP-NANA, and treatment of the LOS with neuraminidase reversed this affect. Gonococci incubated with human PMNs also had decreased expression of the 4.5-kD+ epitopes. A detergent extract of gonococci incorporated radiolabeled NANA in the LOS, suggesting the presence of a sialyltransferase in gonococci. Exogenous sialyltransferases also could use LOS as an acceptor.

A surface polysaccharide forms when gonococci are converted to serum resistance by cytidine 5'-monophospho-N-acetyl neuraminic acid.

A serum-susceptible, guinea-pig chamber-passaged, laboratory strain (BS4 (agar)) of Neisseria gonorrhoeae was converted to serum resistance by incubation with cytidine 5-monophospho-N-acetyl neuraminic acid (CMP-NANA) and examined by electron microscopy after staining with ruthenium-red. In contrast to serum susceptible gonococci incubated without CMP-NANA, the majority (60-70%) of the serum resistant organisms showed a surface accumulation of polysaccharide. This surface polysaccharide was enhanced on all the resistant gonococci after incubation with fresh human serum. Control susceptible gonococci were devoid of the polysaccharide after incubation with heated human serum. Identical results were obtained with a fresh gonococcal isolate which had lost serum resistance on subculture but which, in common with 3 other isolates, was restored to serum resistance by incubation with CMP-NANA.