Characterization and antimicrobial susceptibility of motile aeromonads isolated from freshwater ornamental fish showing signs of septicaemia.

A total of 74 phenotypically identified presumptive motile Aeromonas isolates recovered from septicaemic freshwater ornamental fish in Sri Lanka were genetically characterized by sequencing of rpoD and gyrB genes. rpoD/gyrB phylogeny confirmed only 53 isolates as Aeromonas, among which A. veronii was the predominant species (79.2%), followed by A. hydrophila (7.5%), A. caviae (5.7%), A. jandaei (1.9%), A. dhakensis (3.8%) and A. entero pelogenes (1.9%). The aeromonads confirmed by sequencing were further subjected to 16S rDNA PCR-RFLP which substantiated sequencing results for 83% of isolates. Fingerprinting of A. enteropelogenes (n = 42) using ERIC-PCR revealed no dominant clones, and the majority were genetically distinct. All isolates were screened by PCR for 7 virulence determinant genes (aer, act, ast, alt, fla, ser, exu) and 2 integrase encoding genes (intI1, intI2). Each isolate contained ≥3 of the virulence genes tested for, with a heterogeneous distribution. Of the isolates, 77% harboured the intI1 gene, while none had intI2. In vitro antimicrobial susceptibility testing showed highest resistances towards tetracycline (58.5%) and erythromycin (54.7%). Our results indicate the diverse range of aeromonads that could potentially be associated with motile aeromonad septicaemia in ornamental fish. This is the first isolation of A. dhakensis from a septicaemic ornamental fish since its original description from the same host.

Distribution of lipopolysaccharide core types among avian pathogenic Escherichia coli in relation to the major phylogenetic groups.

Five distinct lipopolysaccharide (LPS) core types, namely R1-R4 and K12 have been identified in Escherichia coli. The aims of this study were to determine, primarily by means of PCR, the distribution of those oligosaccharide core types among avian pathogenic E. coli and their relationship to phylogenetic groups. To identify putative avian pathogenic E. coli, serum resistance and the presence of three virulence genes encoding temperature sensitive haemagglutinin (tsh), increased serum survival (iss) and colicin V (cvaC) were determined. Of the 143 clinical isolates examined 62% possessed the R1 core, 22% were R3, 13% were R4 and 3% were R2. Fifty commensal isolates consisted of 58% with R1 core, 38% with R3 core, 4% with R4 core, and none with R2. None of the isolates were of K12 core type. The distribution of core oligosaccharide types in clinical and commensal isolates were not statistically significant (P=0.51). Three genes, tsh, iss and cvaC were found in E. coli of all four core types. The genes tsh (P<0.001) and iss (P=0.03412) were significantly associated with the R4 core oligosaccharide type. The isolates containing R4 core type LPS were mainly confined to phylogenetic group D. The widespread R1 core type showed less ability to possess virulence genes and 83% were in the phylogenetic group A. Results of this study indicated that E. coli with R1, R2, R3 and R4 were important in causing infections in chickens and further, the E. coli with R4 core type were less common among commensals, possessed more virulence genes and were related to phylogenetic groups pathogenic for poultry.

Production of mouse monoclonal antibodies to Pasteurella multocida type A and the immunological properties of a protective anti-lipopolysaccharide antibody.

Eight monoclonal antibodies (MAbs) were produced from mice immunised with whole cells of heat-killed Pasteurella multocida type A which had been cultured under iron-restricted conditions. The MAbs were selected by an enzyme-linked immunosorbent assay (ELISA) in which the antigen consisted of whole bacteria of the immunising strain. Their reactivity was investigated further by immunoblotting, indirect haemagglutination, a complement-mediated bactericidal assay and passive protection of mice. One of the eight MAbs was shown by immunoblotting to react with lipopolysaccharide (LPS), was bactericidal, and completely protected mice against homologous challenge with 10 LD50 of live bacteria. This MAb was selected for further study. Its reaction with LPS of 17 type-A strains and of single strains of types B, D and E was investigated by immunoblotting. Strains that reacted with the anti-LPS MAb in immunoblots were susceptible to its bactericidal activity and gave high ELISA absorbances. Those that did not react were not susceptible to its bactericidal activity and gave low ELISA readings. The relation between bactericidal activity and ELISA absorbance was highly significant (p less than 0.001). Five of the strongly reacting heterologous strains and one non-reacting strain were selected as challenge organisms in a passive protection experiment: only the mice receiving the reacting strains were protected.

Bactericidal activity in the sera of mice vaccinated with Pasteurella multocida type A.

The susceptibility of Pasteurella multocida to killing by serum and the ability of protective vaccines to stimulate this mechanism of immunity in mice were investigated. P. multocida type of bovine origin was used to prepare a vaccine incorporating heat killed organisms and for homologous infection of mice. Bactericidal capacity and ELISA antibody titres were determined for individual mouse sera. Protection was clearly associated with bactericidal antibodies raised by vaccination. The bactericidal assay may be useful as a rapid, simple screening test of vaccinated mice for functional protective antibody levels.

Acute septicaemic pasteurellosis in Vietnamese pigs.

Sixteen isolates of Pasteurella multocida were cultured from cases diagnosed as acute septicaemic pasteurellosis in Vietnamese pigs. The HSB-PCR assay provided rapid presumptive determination of 10 isolates of P. multocida identified as haemorrhagic septicaemia (HS) causing type B cultures (B:2, B:5, B:2,5). Serological designation using the Carter and Heddleston typing systems confirmed these findings, and identified the six HSB-PCR negative cultures as either A:1, A:3 or D:3,4. Biochemical fermentation and REP-PCR revealed phenotypic and genotypic identity between P. multocida type A:1 isolated from Vietnamese pigs and poultry. Marked homogeneity was also demonstrated among HSB-PCR positive swine isolates, which were shown to possess genotypic identity with P. multocida type B:2 from buffaloes diagnosed with HS.

PCR detection and analysis of Pasteurella multocida from the tonsils of slaughtered pigs in Vietnam.

A total of 36 tonsil swab samples were collected from healthy swine prior to slaughter at the abattoirs in Can tho and Tien giang provinces of Southern Vietnam. The presence of Pasteurella multocida in these samples was detected by the combination of direct cultivation and isolation, mouse inoculation and the polymerase chain reaction (PM-PCR). P. multocida was detected in 16 samples by PCR, with 17 strains ultimately isolated. All samples were negative for serogroup B by HSB-PCR and conventional serotyping, with isolates identified as A:3, D:1 or D:3. In addition, all samples were determined to be negative for the P. multocida toxin (PMT). Characterisation of isolated P. multocida by REP-PCR and biotyping revealed nine distinct REP profiles and seven biotypes among the 17 isolates. Some correlation was seen with P. multocida isolated from a previous Australian outbreak of acute swine pasteurellosis, and those isolated from fowl cholera outbreaks in Vietnamese poultry.

Development of a clinical syndrome resembling haemorrhagic septicaemia in the buffalo following intravenous inoculation of Pasteurella multocida serotype B:2 endotoxin and the role of tumour necrosis factor-alpha.

Clinical changes and acute phase responses, including tumour necrosis factor-alpha (tnfalpha), in six buffalo calves were examined following intravenous inoculation of a bolus of endotoxin (1 microg kg(-1) bodyweight in 10 ml of phosphate-buffered saline [ pbs ]) extracted from Pasteurella multocida serotype B:2, the bacterium responsible for haemorrhagic septicaemia (hs) in Asia. Endotoxin injection caused a rapid onset of clinical signs characterised by dullness, sternal recumbency, elevated rectal temperatures, excessive salivation and dyspnoea that lasted for up to 12 hours post-inoculation (p.i.). Serum concentrations of tnfalpha rose within 1 hour p.i. to reach peak values ranging between 8 and 140 ng ml(-1) at 1-2 hours p.i. and then declined rapidly to baseline levels 3-5 hours p.i. Endotoxin injection induced other acute phase changes, including a rapid leucopenia and reductions in the serum concentrations of iron and zinc and a delayed but prolonged increase in haptoglobin from 12 hours p.i. that reached a plateau from about 60 hours p.i. Three control calves injected with 10 ml pbs showed no clinical or blood compositional changes. By reproducing key signs of hs the work confirms a pivotal role of endotoxin in the pathogenesis of hs and emphasises the exquisite sensitivity of the buffalo to P multocida endotoxin.

Persistence of the carrier status in haemorrhagic septicaemia (Pasteurella multocida serotype 6:B infection) in buffaloes.

Fifty-seven young buffaloes were experimentally infected or naturally exposed to haemorrhagic septicaemia (HS). Of these animals 32 became immune carriers. They were observed in groups for periods up to 360 days to monitor the appearance of pasteurellae in the nasopharynx and antibody status. In most animals pasteurellae appeared in the nasopharynx for a short period initially and disappeared. The organism reappeared intermittently and the longest observed period of reappearance was 215 days after exposure. All animals showed rising antibody titres with a peak lasting for 150 to 180 days and declining thereafter. Pasteurellae persisted in the tonsils and were isolated from 20 out of 27 carriers after slaughter. The longest period when isolation was made after slaughter was 229 days. The organism lodged in the tonsils was unaffected by antibacterial therapy using drugs to which the organism displayed in vitro sensitivity. It is concluded that in HS, carrier animals exist in an active as well as a latent state, the former appearing for short intermittent periods between long latent periods, when pasteurellae continue to remain in the tonsils which constitute a long-term reservoir.

Role of endotoxin in the pathogenesis of haemorrhagic septicaemia in the buffalo.

The pathogenesis of haemorrhagic septicaemia in buffalo infected with Pasteurella multocida is poorly understood. However, the characteristic of sudden onset leading to the rapid death of infected animals is similar to that seen in other clinical conditions known to involve endotoxic shock. The objectives of the work were to assess the contribution of endotoxaemia to the disease's pathogenesis and to characterize the pathophysiological reaction, including the acute phase response, of buffalo to experimental infection with P. multocida serotype B:2, the bacterium responsible for the disease in Asia. After intranasal infection of eight buffaloes with a culture of a field isolate of P. multocida serotype B:2, three animals succumbed to the disease at 26-30 h post-infection (p.i.) and five survived. Rectal temperatures of infected animals rose to a peak at about 10 h p.i. and surviving animals showed a second peak in rectal temperature at 36 h p.i. Endotoxin was present only in serum of non-surviving animals 3-5 h before death or killing during which time concentrations increased rapidly, correlating with the development of overt clinical signs and reductions in rectal temperature, concentrations of white blood cells, serum thyroxine, iron, copper and zinc, an increase in serum haptoglobin and cortisol and the detection of a low-grade bacteraemia. A strong acute phase response was maintained in surviving animals with a progressive rise in serum haptoglobin over 96 h p.ia slow rise in the serum copper concentration from 24 h p.i. and an increase, from about 65 h p.iin serum alpha(1)-acid glycoprotein. The findings demonstrate that a progressive endotoxaemia and associated sequelae correlates with the development of overt haemorrhagic septicaemia disease and sudden death in buffalo.