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.     

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.     

Cloning and characterisation of the Pasteurella multocida ahpA gene responsible for a haemolytic phenotype in Escherichia coli

Haemolysins are membrane-damaging agents which have been described as bacterial virulence factors due to their ability to lyse erythrocytes and other host cells, and therefore inducing a greater inflammatory response (Elliott et al., 1998). Pasteurella multocida was found to be haemolytic under anaerobic conditions. In this study, we cloned and characterised a P. multocida gene, designated ahpA, which conferred a haemolytic phenotype on Escherichia coli when incubated under anaerobic conditions. A deletion was introduced into the ahpA open reading frame which abolished the haemolytic phenotype. The clone containing ahpA showed erythrocyte specificity, causing haemolysis of bovine and equine erythrocytes, and demonstrated weak haemolysis on ovine erythrocytes. Upon further investigation, AhpA was found to affect the expression of the E. coli K-12 latent haemolysin, SheA, under anaerobic conditions.     

The role of Penicillia in ryegrass staggers

Tremorgenic strains of Penicillium verrucosum var cyclopium, P canescens, P janthinellum, P novaezeelandiae and P estinogenum were isolated from the faeces of 15 of 23 affected sheep and cattle in eight of nine field outbreaks of ryegrass staggers. One tremorgenic strain of P griseofulvum was isolated from the faeces of one of 25 sheep grazing in unaffected flocks. Tremorgenic strains of P verrucosum var cyclopium, P canescens, P janthinellum and P estinogenum were also isolated from the A horizon of New Zealand soils. Since a large proportion of experimentally dosed live P verrucosum var cyclopium died during passage through the gut, the faecal evidence from naturally staggering animals suggests that at least some outbreaks of ryegrass staggers are caused by tremorgenic Penicillia and that their source may be soil.     

Isolation of dermatophytes from domestic animals in Norway

The examination of 2066 skin scrapings from domestic animals in the period from July 1981 to June 1984, revealed dermatophytes in 439 samples. Dermatophytes isolated were: M. canis in dog, cat, cattle, horse, swine, goat, rabbit and hamster, M. equinum in dog and horse, M. gypseum in horse, T. equinum in horse and cattle, T. mentagrophytes in dog, cat, cattle, horse, guinea pig and rabbit, T. verrucosum in cattle and E. floccosum in dog.     

Haemolytic activity of sheep complement for two assay systems

Sheep complement (C) is haemolytic for sheep erythrocytes sensitized with rabbit antibody (sheep E-rabbit A) provided serum is used as soon as possible after collection. If left at 4 °C to separate from the clot, serum C activity for sheep E-rabbit A is markedly reduced. Heparinized plasma retains its haemolytic titre for at least 24 h at 4 °C. Plasma from Mg²⁺-ethyleneglycoltetraacetic acid (EGTA) blood is non-haemolytic, but addition of Ca²⁺ partially restores the titre. A high concentration of rabbit A is necessary to sensitize sheep E.Sheep C is haemolytic for human erythrocytes sensitized with sheep antibody (human E-sheep A) in the presence of Mg²⁺-EGTA. This C activity is stable at 4 °C for 24 h in serum, Mg²⁺-EGTA plasma and heparinized plasma. Haemolytic activity of serum heated at 50 °C for 30 min was restored by a factor B containing CM-cellulose fraction of foetal lamb serum in the presence of Mg²⁺-EGTA for human E-sheep A but not sheep E-rabbit A.These findings show that sheep C haemolysis of sheep E-rabbit A requires a Ca²⁺-Mg²⁺-dependent pathway that is labile in vitro for 24 h at 4 °C.     

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.     

Dermatomycosis in sheep caused by a rare strain of Trichophyton verrucosum

In the group of sheep reared for blood collections the enzootic occurrence of dermatomycosis was found affecting the hair on the head and back. From the pathological material obtained from the lesions of twelve animals an identical strain of Trichophyton verrucosum was cultivated which differed from common isolates of this type macromorphologically. By its ability to grow in the medium without vitamin and by its thermotolerance this isolate resembled the physiological variety T. verrucosum Bodin 1902, var. autotrophicum Scott 1976. On the example of trichophytosis in the described sheep flock epizootological importance of foci of this infection is demonstrated in flocks where no immunopreventive precautions are taken.     

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.     

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.     

The diagnosis of swine dysentery and spirochaete diarrhea. 1. Cultural-biochemical differentiation of intestinal Serpulina in routine diagnosis]

Frequent incidence of Serpulina strains showing all cultural and biochemical characteristics of Serpulina (S.) hyodysenteriae except of being indole negative, and alpha-galactosidase positive isolates showing strong haemolysis on Columbia agar with 5% sheep blood and trypticase soy agar with 5% ox blood, respectively, was the cause to evaluate common biochemical and cultural methods in Serpulina routine diagnostics. To this purpose ten type and reference strains as well as 47 field strains were examined for their ability to produce indole, haemolysis, hippurate cleavage, alpha-galactosidase, alpha- and beta-glucosidase activity. Two four-hour identification-systems were used, RapID ANA II and Rosco diagnostic tablets. The ability to produce indole was determined by different methods. All investigations were carried out at least two times. For the investigation of haemolytic patterns trypticase soy agar with 10% ox blood proved to be most effective. Results received using this agar could always be confirmed by the ring phenomenon. Determining the ability to produce indole by adding p-dimethylaminocinnamaldehyde to bacterial growth collected on a cotton swab was confirmed to be more sensitive than other methods. Both four-hour-systems were shown to be useful in Serpulina diagnostics, though in the RapID ANA II only four of 18 available reactions could be used and the hippurate cleavage reaction has to be carried out additionally. Using cultural and biochemical methods, it was possible to assign the type and reference strains to the correct species, as well as 46 of 47 field isolates could be identified including all five known intestinal Serpulina species from swine. 27 strains were determined as S. hyodysenteriae, nine of these isolates atypically being indole negative. In contrast one canine S. pilosicoli strain was atypical showing indole production. Therefore incidence of indole negative variants of S. hyodysenteriae as well as indole positive S. pilosicoli isolates must be taken into consideration.     

Cloning and characterisation of the ahpA gene of Pasteurella multocida serogroup b:2 (strain P52): short communication

Pasteurella multocida B:2 is responsible for haemorrhagic septicaemia in cattle and buffaloes, causing severe economic losses in the developing countries. In the present study, the ahpA gene of P. multocida B:2 (P52) was cloned, sequenced and compared with the previously reported ahpA gene sequence in P. multocida A:1, which is responsible for its haemolytic phenotype. E. coli DH5a cells were further transformed with recombinant plasmid carrying the ahpA gene from P. multocida B:2 (P52) but SDS-PAGE analysis failed to show the expression of haemolysin protein. Slight haemolysis was albeit observed in horse blood agar plates streaked with recombinant E. coli carrying the ahpA gene. Our study indicates that there is 99.6% similarity and 0.4% divergence between ahpA gene of P. multocida B:2 (P52) and P. multocida A: 1, while membrane topology analysis has predicted that ahpA is an inner membrane protein with two strong hydrophobic regions at the N and C terminals. The presence of significant homology in ahpA sequence in A: 1 and B:2 perhaps suggests a common mechanism of pathogenesis in different species of animals.     

Experimental chronic copper toxicity in sheep. Changes that follow the cessation of dosing at the onset of haemolysis.

Eight sheep were given daily oral doses of copper sulphate until haemolysis occurred. Three of the sheep developed further periods of haemolysis after dosing ceased. Serum enzyme and urea levels were measured throughout the experiment and compared to those obtained from three undosed control sheep. Serum enzyme levels rose prior to haemolytic crises and urea levels rose subsequent to haemolysis in animals that died or were killed in extremis. Severe morphological changes were seen in liver, kidney and brain. Tissue levels of copper and iron were markedly elevated. It is concluded that tissue damage continues even after the cessation of ingestion of copper and that the damage can be severe enough to lead to repeated haemolytic crises.     

Effect of Eperythrozoon ovis on the lysis of sheep erythrocytes in the complement fixation test

When erythrocytes from sheep experimentally infected with Eperythrozoon ovis were used in the titration of reagents for a standardised complement fixation test, increased amounts of both haemolysin and complement were required for erythrocyte lysis compared with preinfection titrations. The haemolysin requirement increased by up to 125% at 55 days post-infection and complement requirement increased by up to 40% at 40 days post-infection. These changes appeared to correlate with the development of a macrocytic anaemia in affected sheep rather than E. ovis parasitaemia. The results emphasise the need to carefully monitor the haematological parameters of sheep used as sources of erythrocytes for the complement fixation test.     

Use of erythrocyte fragility profiles for monitoring immune-mediated haemolysis in horses

The fragility of erythrocytes is easily demonstrated by their ability to withstand osmotic swelling and lysis in solutions of increasingly hypotonic saline. In healthy animals a plot of percentage haemolysis against increasing hypotonicity produces a sigmoid curve. Using the same data a derivative curve calculated from haemolytic increments shows a normal distribution of fragility within samples. In enhanced fragility due to immune-mediated haemolytic anaemia, these profiles of haemolysis are markedly altered and the derivative curve becomes multiphasic, indicating the presence of subpopulations of erythrocytes with differing fragility ranges. Analysis of these profiles in a case of intravascular immune-mediated haemolysis in a horse provided useful graphic information for diagnosis and prognosis. In particular, the size of the subpopulation showing increased fragility could be assessed in the acute phase and monitored during recovery.     

Chronic Copper Poisoning in Sheep: Structural Changes in Erythrocytes and Organs

Four cases of experimental copper poisoning in sheep were examined. Light microscopical and ultrastructural alterations of erythrocytes were observed a few hours before a significant haemolysis was evident. Heinz body formation in otherwise unchanged red cells was the first morphological alteration observed. The Heinz bodies were predominantly membrane-attached. During the haemolytic crisis severe erythrocytic distortion, structural membrane alteration and Heinz body-containing ghost cells were observed. Erythrophagocytosis was mainly located to the RE cells of the spleen. Intrafollicular necroses were found in all histological sections from the spleen. Pathological changes in liver and kidney were comparable to those of earlier reports, comprising hepatocellular and renal tubular necrosis.     

Variations in sheep serum conglutinating and haemolytic activity for sheep erythrocytes sensitized by rabbit antibody

Based on conglutinating and haemolytic reactions with sheep erythrocytes (E) sensitized by rabbit antibody (A), three types of sheep sera were encountered. Type 1 sera do not conglutinate or haemolyse sheep E-rabbit A. Type 2 sera failed to conglutinate, but are haemolytically active. Type 3 sera have both activities. Serum from one type 1 sheep still failed to conglutinate 5 days after venepuncture but was now haemolytically active (i.e., type 2). Some sheep that initially had type 2 sera had, five days after an intraperitoneal injection of yeast cells, sera with conglutinating activity (type 3 sera). Type 1, 2 and 3 sera all had haemolytic activity with human E-sheep A indicator cells.Pooled type 3 sera have the highest conglutinating titres with sheep E-rabbit A after 10 min incubation at 39 °C. At this stage, the haemolytic titres were very low. From 10 min, the conglutinating titres decreased whereas the haemolytic titres gradually increased until 80 min. Optimal conglutinating activity required less rabbit A to sensitize sheep E than did haemolytic activity.     

Comparison of necrotoxigenic Escherichia coli isolates from farm animals and from humans

Necrotoxigenic Escherichia coli (NTEC) isolated from animals and humans can belong to the same serogroups/types and produce or carry the genes coding for fimbrial and afimbrial adhesins of the same family, P, S, F17, and/or AFA, raising the question of a potential zoonotic source of human infection. The main purpose of this study was to compare 239 NTEC1 strains (45 from cattle, 65 from humans and 129 from piglets) and 98 NTEC2 strains from cattle, using a uniform and standardized typing scheme. The O serogroups and the biotypes recognized amongst NTEC1 and NTEC2 strains were quite varied, although some were more frequently observed (serogroups O2, O4, O6, O8, O18, O78, and O83 and biotypes 1, 2, 5, 6, and 9). Hybridization, results with gene probes for the P family (PAP probe), S family (SFA probe), AFA family (AFA probe), F17 family (F17 probe) of fimbrial and afimbrial adhesins, could differentiate most NTEC1 strains, which are PAP-, SFA- and/or AFA-positive, from NTEC2 strains, which are mainly F17- and/or AFA-positive, but were of no help in differentiating between NTEC1 strains from cattle, humans, and piglets. All but seven (98%) NTEC1 and NTEC2 strains were serum resistant, 199 (59%) produced an aerobactin, and colicin (I, V, or unidentified) was produced by 22-34% of them. On the other hand, more than 90% of the NTEC1 strains were haemolytic on sheep blood agar compared with only 40% of the NTEC2 strains. Production of a classical haemolysin, active on sheep erythrocytes, and hybridization with the PAP probe were associated in a majority of NTEC1 strains (63-81%), but very rarely in NTEC2 strains (3%). Production of enterohaemolysin and hybridization with the PAP probe were much less frequently associated in NTEC strains (1-9%). It was thus possible neither to completely differentiate NTEC1 strains from cattle, humans, and pigs, nor to define a signature for the NTEC strains. Necrotoxigenic E. coli must still be identified on the basis of the production of the Cytotoxic Necrotizing Factors 1 or 2 (or of their encoding genes) and complete differentiation of NTEC1 strains from cattle, humans, and piglets, use additionnal methods.     

An outbreak of ringworm in sheep in Ireland caused by Trichophyton verrucosum

An outbreak of ringworm in sheep in Ireland caused by Trichophyton verrucosum is described. The flock consisted of 110 sheep and two separate groups within the flock were affected. Eleven of the first group of 23 sheep and five of the second group of 25 sheep showed lesions. Contact with infected cattle and fomites contaminated by the cattle are believed to have caused the outbreak. An excellent response was obtained in both infected groups by treatment with griseofulvin at a dose rate of 7.5 mg/kg daily for seven days.     

Serological reactions in sheep and cattle experimentally infected with three Australian isolates of bluetongue virus

Serums from 103 sheep and 24 cattle experimentally infected with one of 3 serotypes of bluetongue virus isolated in Australia were tested for antibody to bluetongue virus in the serum neutralisation test and the agar gel diffusion precipitin test. Antibody to bluetongue virus was first detected by these tests 8 to 10 days after intravenous infection in 4 sheep that were bled daily for serum analysis. The agar gel diffusion test failed to detect antibody in 28% (29/103) of sheep which had seroconverted in the serum neutralisation test. A further 7% (7/103) of sheep serums were negative in both tests 14 to 22 d after infection. Both tests detected antibody to bluetongue virus in all cattle serums by 10 days after detection of viraemia. In comparison with the intravenous route of infection, extended prepatent periods for the commencement of viraemia resulting from intradermal, subcutaneous and intrauterine routes of infection in the cattle caused corresponding delays in the detection of antibody. For example, one cow that was infected by intrauterine inoculation did not become viraemic until 22 d after inoculation and antibody was not detected until 32 d after inoculation.