Haemolytic and cytotoxic activities of the Tween 80-extracted putative haemolysin of Pasteurella multocida B:2

The objective of this study was to investigate the haemolytic and cytotoxic activity of Pasteurella multocida B:2 strains, originally from cases of haemorrhagic septicaemia in cattle. All six P. multocida B:2 strains were non-haemolytic on sheep blood agar (SBA) and horse blood agar (HBA) when grown aerobically and on SBA anaerobically but they were haemolytic on HBA when grown anaerobically. No haemolytic activity against horse red blood cells was detected in culture supernates from aerobically or anaerobically grown cultures and only very weak haemolytic activity was obtained in supernates or pellet fractions from sonicated cells. However, after repeated extraction of sonicated cells with Tween 80, haemolytic activity was found in various cell fractions, both Tween-soluble and -insoluble. The Tween-extracted putative haemolysin and other bacterial fractions were also cytotoxic for mouse macrophage-like J774.2 cells. Further characterisation of the putative haemolysin revealed it to be a heat-labile, non-pore-forming protein of molecular weight >10 kDa whose activity was completely destroyed by trypsin and greatly reduced with protease and proteinase K treatment. Congo red also reduced the haemolytic activity. Non-denaturing gel-electrophoresis and RBC agar overlay revealed clear haemolytic zones but suggested that Tween was bound to some component of the P. multocida B:2 fractions and was responsible, to some extent, for the haemolytic activity observed. However, the effect of heat and other reagents on the Tween-extracted fractions and the lack of haemolytic activity in different Tween-extracted cell fractions of organisms other than P. multocida suggested that some proteinaceous component of the organism could indeed act as a haemolysin. This putative haemolysin may be one of the virulence attributes of P. multocida, but its characterisation and role in pathogenesis require further study.     

Isolation and characterization of a haemolysin from Trichophyton mentagrophytes

Haemolytic activities of Trichophyton (T.) mentagrophytes were detected and characterized by qualitative and quantitative assays. On Columbia agar supplemented with blood from horses, cattle or sheep, T. mentagrophytes expressed a strong zone of complete haemolysis. No haemolytic activities could be detected in the closely related T. verrucosum var. ochraceum. The same results were obtained after cultivation of the fungi on sterile cellulose acetate filters placed on the surface on Columbia blood agar. After removal of the filter, complete haemolysis was detected below the colony of T. mentagrophytes. A soluble haemolysin from culture supernatant of this strain was isolated and partially purified. Specific haemolytic activity per mg protein was enriched 2.6-fold in filtrate F(1), a fraction obtained as filtrate after filtration through 3kDa cut-off membranes. The partially purified haemolysin was neither affected by proteinase K treatment, nor by high and low temperatures, suggesting that it represents a small peptide haemolysin. Accordingly, in a commercial enzymatic activity test only the crude culture filtrate, but none of the subsequent purification fractions showed reactivity. Evaluation of the specificity of the haemolysin using erythrocytes from different mammalian species revealed that sensitivity was highest to those of equines, followed by erythrocytes from sheep, cattle, swine, dogs and humans. None of the erythrocytes was lysed by filtrate F(1) from T. verrucosum var. ochraceum. Furthermore, different eukaryotic cell lines from different species were tested in their sensitivity to cytolytic activities of the haemolysin, but no membrane damage could be detected.     

Endothelial cytotoxicity of Actinobacillus pleuropneumoniae

The cytotoxicity of Actinobacillus pleuropneumoniae serotype 1 strain CM5 for porcine and bovine endothelial cells in vitro, was dose-dependent. This strain and its attenuated and avirulent substrain CM5A were equally cytotoxic. The cytotoxicity observed during five hours of exposure of endothelial cells to bacterial products was abolished if the bacteria were inactivated by heat or sonication. Exposure of the endothelial cells for five hours to 100 and 200 micrograms of purified lipopolysaccharide resulted in a partial cytotoxicity only, which was not enhanced in the presence of fresh guinea pig serum. The cytotoxicity of viable bacteria could be neutralised by a polyclonal rabbit antiserum to the purified 104kD haemolysin. A bacteria-free supernate of a culture of strain CM5 had both haemolytic and cytotoxic activity. The haemolytic activity could be neutralised completely by the anti-serum to the 104kD haemolysin, whereas the cytotoxic activity was only partially neutralisable. Hence A pleuropneumoniae is cytotoxic for endothelial cells and this cytotoxicity is possibly mediated by the 104kD haemolysin.     

Identification of a new Escherichia coli She haemolysin homolog in avian E. coli

Haemolysin is one type of virulence factor that assists in the pathogenesis of Escherichia coli. Currently, hemolytic activity in E. coli has been attributed to haemolysin genes found in either uropathogenic or enterohemorrhagic E. coli. Both haemolysins are classified as RTX toxins because they both have repeats in toxin domains and share similar operon organization, sequence homology, and mechanisms of action. Haemolytic avian E. coli isolates, however, lack either E. coli haemolysin gene. To investigate the avian E. coli haemolysin, a genomic library was made from an avian pathogenic E. coli. A haemolytic clone that was isolated was shown to contain homology with sheA, an E. coli K- 12 gene which causes haemolysis when present in high copy number. The cloned haemolysin gene, hlyE, lacked the conserved amino acid sequence and accessory genes common to all RTX toxins. DNA hybridizations and polymerase chain reaction amplifications showed that the nucleotide sequences homologous to hlyE were not present in a collection of three O157: H7 E. coli, five haemolytic canine uropathogenic E. coli, one haemolytic O26 E. coli, and three haemolytic avian pathogenic E. coli. Thus we have identified a new E. coli haemolysin distinct from the RTX haemolysins and have shown that some avian pathogenic E. coli possess a haemolysin with no apparent homology to hlyE or RTX haemolysins.     

Vaccination against experimental staphylococcal mastitis in ewes

Experiments were carried out in ewes using a new vaccine developed for the prevention of mastitis caused by Staphylococcus aureus. The vaccine comprised three major components: (i) killed S aureus cells which had been cultured to induce synthesis of pseudocapsule; (ii) toxoided staphylococcal beta haemolysin and (iii) the adjuvant dextran sulphate. Ewes systemically vaccinated twice during pregnancy developed significantly elevated circulating levels of IgG1 and IgG2 anti-pseudocapsule antibody, as well as increased serum titres of anti-beta haemolysin. Five different strains of S aureus were used to challenge both vaccinated and control ewes by the intramammary route during the ensuing lactation. The incidence of acute gangrenous mastitis and nonacute, clinical mastitis was significantly lower in vaccinated than in control groups after challenge with each strain. Vaccinated ewes produced significantly more milk than control ewes after challenge with four of the five strains of S aureus.     

Studies on activation and levels of haemolytic complement of buffalo (Bubalus bubalis). 1. Classical complement pathway

Optimum conditions for haemolytic complement (HC) assay in buffalo serum were standardized. In all, 11 indicator systems of red blood cells (RBC) and haemolysins were investigated. Maximum HC CH50 titre was obtained with rabbit RBC sensitized with goat haemolysin. The effect of pH, Ca2+ and Mg2+ concentration, ionic strength, time and temperature were studied. Of all the variables, ionic strength influenced the HC activity most significantly. The standard system for titrating the HC consisted of rabbit RBC sensitized with goat haemolysin, sucrose-veronal buffer with pH 7.5, ionic strength 0.023 M and Ca2+ and Mg2+ concentrations 6 x 10(-4) and 2 x 10(-3) M, respectively. Incubation at 37 degrees C for 2 h gave highest haemolytic activity. With this protocol 5-7-fold higher HC activity was recorded than with prestandardized conditions. Levels of HC were determined in the sera of 98 buffaloes aged from 1 month to 12 years. The lowest mean CH50 units of 401 +/- 0.35 per ml were recorded in buffalo calves below 3 months of age. The mean HC levels increased with age, reaching peak values of 2349 +/- 62.25 CH50 units/ml in 2-3-year-old buffalo. Animals in the age group 5-12 years had significantly decreased (P less than 0.05) mean HC levels of 1545 +/- 68.94.     

Demonstration of Alternative and Classical Complement Pathway Activity in Colostrum from Buffalo (Bubalus bubalis)

Buffalo colostrum caused lysis of unsensitized red blood cells (RBC) from sheep, goats, rabbits and chickens. RBC from cattle and buffalo were resistant to lysis. That lysis was due to the presence of natural antibodies to these RBC was ruled out since there was no reduction in haemolytic titres even after adsorption with the respective RBC. The addition of EGTA to the diluent had no effect on the haemolytic activity. These findings indicate the presence of alternative complement pathway (ACP) activity in buffalo colostrum. The haemolytic activity of buffalo complement for unsensitized rabbit RBC was reduced to very low levels by heating at 50°C for 45 min. Treatment with zymosan also inhibited the haemolytic activity, while inulin had no effect. The maximum activity of ACP occurred in the presence of 4 mmol/L Mg²⁺ in the diluent. The range of ACP activities in colostrum from buffaloes varied from 4.06 to 8.48 CH₅₀ units/ml. Using a standard system for titrating the classical complement pathway and rabbit red blood cells sensitized with goat haemolysin, the range of complement activity in buffalo colostrum was 4.81–6.77 CH₅₀/ml.     

Cultural characteristics and virulence of strains of Fusobacterium necrophorum isolated from the feet of cattle and sheep

Sixty-one isolates of Fusobacterium necrophorum were recovered for study. Thirty-one were obtained from lesions of foot abscess in cattle (25) and sheep (6), 28 were from interdigital lesions in cattle and 2 were from the normal interdigital skin of cattle. The majority of isolates from lesions of foot abscess were virulent, belonged to biotype AB (Fievez 1963), produced flat, irregular shaped, greyish colonies and haemolysis on blood agar, and grew as turbid filamentous suspensions in liquid media. They produced a soluble exotoxin, a leucocidin, and were pathogenic for cattle and mice. Virulent isolates also produced a haemolysin which most readily lysed bovine, equine and chicken erythrocytes; those from sheep were less susceptible while those of rabbit and pig were the most resistant. Isolates recovered from lesions of the feet not classified as foot abscess and from clinically normal feet were predominantly of the B biotype and caused few experimental lesions, produced convex, round, yellow colonies, flocculated and sedimented while growing in liquid medium and produced little or no haemolysin or leucocidin. Routine differentiation between virulent and non-virulent bovine isolates of F. necrophorum could be achieved by assessing the colour, morphology, and degree of haemolytic activity of colonies grown on blood agar.     

Studies on some biochemical characteristics of Staphylococcus aureus of buffalo mammary origin

A total of 49 isolates of Staphylococcus aureus of buffalo mammary origin were studied for biochemical characteristics. Coagulase production, clumping factor, haemolytic activity, pigment production and fermentation of maltose and mannitol were employed to differentiate S. aureus from S. hyicus and S. intermedius. Out of 49 isolates, 97.95, 93.87, 93.87, 89.79, 95.91, 100.0, 95.91, 59.18, 100.0, 100.0, 100.0, 89.79, 91.83 and 100.0% isolates were positive for coagulase production, protein-A production, haemolysin production, thermostable nuclease production, deoxyribonuclease production, tellurite reduction, nitrate reduction, lipase production, phosphatase production, mannitol fermentation, glucose fermentation, M.R. test, V.P. test and pigment production respectively. The only isolate from which coagulase production could not be detected, however, showed haemolytic activity, protein-A productivity, pigmentation and mannitol fermentation. One of the protein-A negative isolate was coagulase positive and showed mennitol fermentation, pigmentation and haemolytic activity. The study revealed that the biochemical characteristics of S. aureus of buffalo mammary origin did not differ from those of cattle origin. Coagulase, haemolysin, thermostable nuclease, deoxyribonuclease, phosphatase, lipase, tellurite and nitrate reduction closely related with protein-A. The presence of protein-A seems to be as reliable an indicator for S. aureus of buffalo origin as is coagulase production.     

Development and evaluation of an enzyme-linked immunosorbent assay for bovine antibody (IgG) to Pasteurella haemolytica

The sensitivity of an indirect enzyme-linked immunosorbent assay (ELISA) for bovine IgG serum antibody to Pasteurella haemolytica was compared with that of an indirect hemagglutination (IHA) test. Pasteurella haemolytica serotypes were grown in a chemically defined cell culture medium, and soluble antigens released into the growth medium were used in the ELISA and IHA test. An ELISA with serotype-1 antigen consistently detected antibody in sera that were positive by IHA test (correlation, 99%). Sera reacting with serotype-1 ELISA antigens also reacted with ELISA antigens prepared from other serotypes. Although ELISA titers averaged 5 log2 units higher than IHA titers, plots of titers determined by the 2 methods were approximately linear. Titer increases detected in paired serum samples by either test were similar. The ELISA was more sensitive than was the IHA in detecting colostral IgG antibody in serum of newborn calves. The ELISA uses a simple, stable antigen preparation and detects antibody to P haemolytica serotypes that commonly infect cattle.     

Characterisation of Escherichia coli strains associated with canine urinary tract infections

Of 33 Escherichia coli strains isolated from canine urinary tract infections, 22 were haemolytic and 27 were classified into O serogroups, the most common being O4, O6, O2 and O83. P-fimbriated strains were haemolytic and belonged mainly to serogroups O4 and O6. Twenty-nine strains possessed type-1 fimbriae but only small numbers possessed S fimbriae, type-1C fimbriae, X adhesins or the aerobactin system. It is postulated that P fimbriae and haemolysin production contribute to bacterial virulence in canine pyelonephritis and cystitis.     

Changes in LC50 in an in vitro egg development assay during the patent period of Haemonchus contortus in sheep

Sera of various species inhibit the activity of haemolysin produced by Treponema hyodysenteriae. Low density and high density lipoprotein fractions from swine and human sera suppressed the haemolysin, with no single class of lipoproteins in swine serum showing predominant activity. Albumin, immunoglobulin G or blood sugars in swine serum showed no inhibitory activities.     

In vitro efficacy and pharmacodynamic profiles of four polyether ionophores against methicillin-resistant Staphylococcus spp.

The objective of this study was to determine the minimum inhibitory concentrations (MICs) and pharmacodynamic profiles of four ionophores (lasalocid, monensin, narasin and salinomycin) against staphylococcal isolates from clinical cases of human and veterinary staphylococcal infections, and to determine the effect of methicillin resistance on the antimicrobial activity of ionophores. Broth microdilution MIC testing was used to determine antimicrobial activity against 156 staphylococcal isolates of human and veterinary origin. Pharmacodynamic profiles were examined using time-kill kinetics profiles against an ATCC type strain of Staphylococcus aureus and a clinical isolate of methicillin-resistant Staphylococcus pseudintermedius. All tests were performed in accordance with CLSI guidelines. All four ionophores demonstrated antimicrobial activity against methicillin-resistant staphylococci at concentrations similar to those observed for methicillin-susceptible isolates of the same species. Testing of human and veterinary MRSA isolates also showed that MIC values were not influenced by the host origin of the isolates. Pharmacodynamic profiles were similar for both isolates tested across all four ionophores, with similar reductions in viable cell counts being observed over an 18- to 24-hr period. Lasalocid, monensin, narasin and salinomycin all demonstrated antimicrobial activity against staphylococcal isolates of human and veterinary origins, with activity being unaffected by methicillin resistance status, although some Staphylococcus species-specific effects were observed that require further investigation.     

Modulation of IgG subclass expression during antibody responses in sheep

Experiments were undertaken to investigate IgG subclass expression during epitope-specific antibody responses in sheep. Animals were immunised with conjugates of bovine serum albumin-DNP (BSA-DNP), or killed Staphylococcus aureus-DNP (Sa-DNP) alone or with muramyl dipeptide (MDP), dextran sulphate (DXS), or staphylococcal exotoxins (toxin). Sheep received two injections of the same preparation intracutaneously at six weeks interval. Total and IgG subclass-specific anti-DNP, and anti-carrier (BSA or S aureus) antibody levels were measured by ELISA in blood taken at weekly intervals before and after immunisation. Anti-DNP antibody levels in animals given Sa-DNP alone were considerably greater than in those immunised with BSA-DNP alone. Toxin suppressed antibody responses to DNP and both carriers; MDP suppressed anti-hapten antibody responses below the levels obtained with antigen alone. Neither toxin nor MDP significantly altered the IgG subclass profile of antibody to DNP bound to either carrier. DXS did not significantly change total levels of anti-DNP antibody measured in sheep given BSA-DNP or S aureus-DNP. However, DXS promoted IgG1 and suppressed IgG2 anti-DNP antibody responses during the secondary response to Sa-DNP but not to BSA-DNP.     

Detection of antibodies to Actinobacillus pleuropneumoniae serotype 12 in pig serum using a blocking enzyme-linked immunosorbent assay

The object was to examine the geographical variation in the presence of superantigenic exotoxins and beta-hemolysin among epidemiologically independent Staphylococcus aureus isolates from bovine mastitis. A total of 462 S. aureus isolates from nine European countries and USA were examined for the presence of genes encoding staphylococcal enterotoxins A-E, and H, toxic shock toxin-1 (TSST-1), and beta-hemolysin, and 128 of these were examined for exfoliative toxins A and B. The detection was done by PCR. Phenotypic methods were used to confirm the PCR-results. None of the 128 isolates carried the genes for exfoliative toxin A or B. The total proportion of isolates in which superantigenic exotoxins were detected varied from 2% (one isolate) of the Danish isolates to 65% (32 isolates) of the Norwegian isolates. This marked and highly significant geographical variation was also present for the individual exotoxins. The genes encoding enterotoxin C, TSST-1, and enterotoxin D were the most common superantigens. The present and earlier studies demonstrate that the superantigenic exotoxins that were investigated in this study, do not play a role in the pathogenesis of bovine S. aureus mastitis. In contrast to the geographical variation among superantigenic exotoxins, 97% of the isolates were PCR-positive for and/or produced beta-hemolysin on 5% calf blood agar. Except for three isolates, the Norwegian isolates were PCR-negative, but positive on 5% calf blood agar. Sequence variation in the primer regions in the beta-hemolysin encoding gene of the Norwegian isolates is suggested, and should be investigated further. The consistent presence of beta-hemolysin suggests that this factor, or a co-existing gene correlated to beta-hemolysin, may be an active virulence factor in the pathogenesis of bovine S. aureus mastitis.     

Cytotoxic activity of Staphylococcus hyicus

Culture supernatants from a number of Staphylococcus hyicus strains caused toxic effects to both murine fibroblast and porcine keratinocyte cells in culture. The extent of cytotoxicity was shown to differ between strains and may provide an indication of strain virulence. Purification of cytotoxic activity produced by S. hyicus (strain P119) using preparative isoelectric-focussing demonstrated it to be cytolytic, haemolytic and non-proteolytic. The cytotoxin demonstrates certain properties in common with the delta haemolysin of Staphylococcus aureus.     

Characterization and immunogenicity of an apxIA mutant of Actinobacillus pleuropneumoniae

Actinobacillus pleuropneumoniae is the aetiological agent of porcine pleuropneumonia, a highly contagious and often fatal disease. A candidate live vaccine strain, potentially capable of cross-serovar protection, was constructed by deleting the section of the apxIA gene coding for the C-terminal segment of ApxI toxin of the A. pleuropneumoniae serovar 10 reference strain (D13039) and inserting a chloramphenicol resistance gene cassette. The mutant strain (termed D13039A(-)Chl(r)) produced an approximately 48kDa protein corresponding to the N-terminus of the ApxI toxin, and exhibited no haemolytic activity and lower virulence in mice compared with the parental strain. The mutant was evaluated in a vaccination-challenge trial in which pigs were given two intra-nasal doses of the mutant at 14 days intervals and then challenged 14 days after the last vaccination with either A. pleuropneumoniae serovar 1 (4074) or serovar 2 (S1536) or serovar 10 (D13039) reference strains. The haemolysin neutralisation titres of the pre-challenge sera were significantly higher in the vaccinated pigs than in the unvaccinated pigs. The mortalities, clinical signs and lung lesion scores in the vaccinated pigs were significantly lower than those in the unvaccinated pigs for the serovar 1 challenge. A significantly lower lung lesion score was also observed in the vaccinated pigs, compared with unvaccinated pigs, for serovar 2 challenge. Our work suggests that the mutant strain offers potential as a live attenuated pleuropneumonia vaccine that can provide cross-serovar protection.     

Effects of Thymus vulgaris L. as feed additive in piglets and against haemolytic E. coli in vitro

The aim of the study was to test Thymi herba (1.66% v/w essential oil with 39% p-cymene and 32% thymol) in the rearing period of piglets as feed additive. Therefore, two feeding trials were performed with piglet groups ranging from 17 to 22 animals each. Either 10 g of Thymi herba/kg feed (Thymi herba group), 10 mg flavophospholipol/kg feed (flavophospholipol group) or nothing (control group) was added to the animals' feed. No significant differences in the performance parameter daily weight gain among any groups were recorded. No differences concerning feed efficiency or isolation of haemolytic E. coli serotypes were shown. In addition, the antibacterial activity of the essential oil of Thymi herba against 39 haemolytic E. coli isolates from the same weaners was investigated in vitro by disk diffusion, minimum inhibitory concentration and bactericidal concentration testing. In contrast to the feeding results, the essential oil of the thyme batch fed showed antibacterial activity against all haemolytic E. coli investigated. This interesting antibacterial potential of Thymi herba prompts further investigations as to its value as feed additive.     

Evaluation of an indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to the Apx toxins of Actinobacillus pleuropneumoniae

The reference strains of the 12 serotypes of Actinobacillus pleuropneumoniae express one or two of three different RTX exotoxins designated Apx I, Apx II and Apx III. The toxins are important virulence factors. In the present study, ELISAs with purified Apx I, Apx II and Apx III, respectively, as antigen were evaluated as candidates for serological diagnosis of Actinobacillus pleuropneumoniae infection in pigs. The pigs were inoculated with biotype 1, serotypes 1-12, and biotype 2, serotype 14, respectively. A strong humoral antibody response was seen to all the three antigens in most pigs irrespective of the serotype used for inoculation. However, titers to the exotoxins secreted by the serotype used for inoculation were generally highest. The results show that toxin proteins of Actinobacillus pleuropneumoniae are antigenically related and that a correlation between serotype and secretion of exotoxin is not revealed serologically in the ELISA test.     

Studies on Aspergillus Fumigatus; Hydrolysis of Polyamino Acids by Mycelial Filtrate and Chromatographic Fractionation of the Filtrate

Mycelial filtrates from Aspergillus fumigatus (AF), shown to possess haemolytic, toxic, casein precipitating, and protein hydrolyzing activity, hydrolyzed poly-L-lysine and poly-L-glutamine in the pH range 4.6—5.3. Incipient activity against poly-L-lysin was observed at pH 9. Owing to spontaneous hydrolysis of the polyamino acid at pH > 10, no activity optimum could be traced.Gel filtration of mycelial filtrate on Sephadex G-75 or G-100 columns offered no definite information whether the protein hydrolyzing activity, using haemoglobin as substrate, at the optimum pH values, 2.9, 4.6 and 10, shows the activity of a single enzyme with more than 1 pH optimum or of more than 1 enzyme active at different pH values. Certain results of the investigations seem to indicate that the protein hydrolyzing activity at pH 2.9 was not caused by enzymes identical with the enzyme (s) causing the protein hydrolyzing activity at pH 4.6 or pH 10.Casein precipitating and protein hydrolyzing activity occurred, following gel filtration on a Sephadex G-100 column, in identical fractions whereas neither haemolysin nor toxin could be found in samples of 0.5 ml fraction solution from any of the fractions after filtration on Sephadex G-75 or G-100 columns.By using antiserum to a crude filtrate from a homologous AF strain it could be shown, applying immuno-electrophoresis, that dialyzed mycelial filtrate contained 8 precipitating antigens whereas proteinase purified by gel filtration and displaying protein hydrolyzing activity at pH 2.9, pH 4.6 and pH 10 contained 4 such antigens.