Auxotrophic, plasmid-cured Salmonella enterica serovar typhimurium for use as a live vaccine in calves.

Calves were vaccinated orally, subcutaneously or intraperitoneally with a smooth, plasmid-cured strain of Salmonella enterica serovar typhimurium, strain 81. Oral vaccination was not effective, as only 1/5 calves survived challenge with virulent S. typhimurium. Strain 81 was attenuated for calves, as only a slight rise in rectal temperatures was detected after vaccination. The organism was excreted by some calves in the faeces, but no signs of diarrhoea were observed after vaccination. After parenteral vaccination, strain 81 was able to reach the intestines, gastric associated lymphoid tissues and other internal lymphoid tissues and remained viable for up to 14 days in the bovine host. After oral challenge with a virulent strain, 9/10 vaccinated calves survived challenge as opposed to 4/10 control calves (p<0.5). Diarrhoea was present in all calves of the control groups, but in only 4/10 of the vaccinated calves. The clinical reactions of the vaccinated calves were milder than in the control calves, as the rises in rectal temperatures were lower, diarrhoea was less severe, and the challenge strain was present in fewer organs from vaccinated calves than control calves. This study showed that parenterally administered Salmonella vaccines can induce both mucosal and systemic immunity, and it is postulated that this capability of strain 81 is related to its colonisation of lymphoid tissues and other systemic and intestinal tissues. This study confirmed that plasmid-cured strains were attenuated in the bovine host and conferred significant protection after parenteral vaccination, but not oral vaccination.

Characterization of south african isolates of Salmonella enteritidis by phage typing, numerical analysis of RAPD-PCR banding patterns and plasmid profiles.

Eleven of the 33 strains of Salmonella enteritidis (S.E.) included in this study belonged to phage type 34. Six strains belonged to phage type 14, six strains to phage type 4 and four strains to phage type 7. The remaining six strains belonged to phage types 35, 1, 24var (a variation of phage type 24), 9a, 1b and an unknown phage type. The majority of S.E. phage type 34 strains (eight of the 11) grouped at R2 > or =0.45 into one RAPD-PCR cluster with two strains of phage types 4, a strain of phage type 24var and a strain of phage type 9a, indicating that they consist of a genetically heterogeneous collection of strains. Two of the remaining three phage type 34 strains grouped into two different clusters, well separated from the other phage type 34 strains. One strain of phage type 34 was genetically diverse and did not cluster with any of the strains included in this study. Three of the phage type 14 strains grouped into cluster 11 at R2 > or =0.72, suggesting that they are genetically closely related. However, the remaining three strains of phage type 14 grouped into two separate clusters. Strains of phage types 7, 35, and 1 grouped in one cluster at R2 > or = 0.55. Our results clearly indicated that S.E. strains of the same phage type are not always genetically related. On the other hand, strains of a high genetic relatedness classified as different phage types. No specific plasmid profile could be linked to any of the phage types. Based on results obtained by LD50 virulence tests, strains containing the 38 MDa plasmid are more virulent compared to strains which do not contain the plasmid.

Phage types of Salmonella enterica serovar Enteritidis isolated in South Africa from 1991-1995.

A total of 615 strains of Salmonella enterica serovar Enteritidis (SE), received from 1991-1995 at the Onderstepoort Veterinary Institute (OVI), were phage typed. Most SE isolates (54,7%) originated from poultry followed by humans (28,5 %) and poultry eggs (9,6 %). Phage type 34 was the most prevalent (40,5 %) of all isolates, followed by phage type 4 (33,8 %). Other phage types identified were 1, lb, 4a, 7, 7a, 9a, 14, 24, 24var and 35 (in total 2,4% of isolates). Most isolates of SE were received from the Western Cape Province (47,4%) and Gauteng (22,3%). In poultry phage type 4 was dominant, but in humans, eggs, goats, ducks, sheep, pigs and rabbits, phage type 34 was the dominant type. It appeared as if the poultry-associated epidemic of SE in South Africa that occurred from 1991-1995 originated in the Western Cape Province during 1991 amongst poultry and then spread from there to humans and eggs and then to the rest of the country, where it emerged during 1993. Results indicate that phage type 34 was the dominant phage type from 1991-1993, but during 1994-1995 its presence declined. During this latter period the presence of phage type 4 increased. This may suggest that two smaller epidemics consisting of the two different phage types might have been responsible for the epidemic that occurred from 1991-1995.

The production of an auxotrophic marked, plasmid-cured Salmonella ser. Typhimurium as a live attenuated vaccine.

A number of amino acid requiring auxotrophic strains of Salmonella Typhimurium were produced by chemical mutagenesis. One of them, strain 81, was cured of the virulence plasmid and attenuated for mice. This strain had an auxotrophic requirement for serine, which could be used as a marker for the differentiation of the vaccine strain from other isolates in the field. The strain still contained the smooth form of the O-antigen, was resistant to Complement-mediated killing of serum and produced type 1 fimbriae. Of the six auxotrophic mutants only this mutant differed in its outer membrane protein profile from that of the parent strain in that an outer membrane protein of about 30 kDa was absent. With the use of the polymerase chain reaction, using total DNA of the cell as template, and with primers targeted to the virulence plasmid, it was shown that the virulence plasmid of Salmonella Typhimurium was completely cured from this strain. This strain also had a LD50-value of 4 log units lower for mice than the parent strain. The plasmid-cured strain gave a very high degree of protection to mice after systemic immunization, but not after oral vaccination. Compared to the parent, strain 81 also had a lower multiplication rate in the liver and spleen after intraperitoneal inoculation, characteristics that could be attributed to plasmid-loss, and it could also not be recovered from the spleen and liver of orally inoculated mice.

Salmonella isolated from crocodiles and other reptiles during the period 1985-1994 in South Africa.

Over a 10-year period, 173 isolates of Salmonella were obtained during routine isolation from reptiles. Of the 173 isolates, 92 different Salmonella serovars were identified. Of them, 61 (66%) belonged to subspecies I, nine to subspecies II and 21 to subspecies III (IIIa and IIIb), and one to subspecies IV. The majority of isolates were from farmed Nile crocodiles (145), three from wild-caught African dwarf crocodiles, 11 from captive snakes, 13 from lizards and one from a tortoise. The isolates from the tortoise and lizards were subspecies I isolates (Zaire and Tsevie, respectively). Of the snakes, nine isolates were S.III. The serovars isolated most often from the crocodiles were of subspecies I (32 serovars). Eight were from subspecies II, seven from subspecies III and one from subspecies IV. The most frequently identified serovars were Typhimurium (seven), Tsevie (six), Duval (six), Schwerin (six), Tinda (six), and Tallahassee (six). On two commercial crocodile breeding farms that had experienced ongoing problems for about two years, many isolates of Salmonella were made. Some of these serovars were isolated more than once, and also months apart. No single Salmonella serovar predominated, nor did a single pathological condition. These salmonellas were predominantly of subspecies I.

First reported African case of Anaerobiospirillum succiniciproducens septicemia.

The first reported African case of septicemia caused by Anaerobiospirillum succiniciproducens is presented. The source of the infection was not identified. The clinical presentation and relative risk factors of the infection, and biochemical characteristics and antimicrobial susceptibility of the organism are described. Recommendations on appropriate antimicrobial therapy and postinfection sequelae are discussed.

The prevalence of intestinal Salmonella infection in horses submitted for necropsy.

Specimens from the ileum, colon and rectum were aseptically collected from 50 consecutive horse carcases submitted for necropsy to the Department of Pathology, Faculty of Veterinary Science, University of Pretoria. These were bacteriologically examined for the presence of Salmonella. Seventeen of these were positive for Salmonella at one or more sites. Serotyping of the isolates revealed a dominance of Salmonella Hayindogo in these horses.

First isolation of Campylobacter jejuni from the vaginal discharge of three bitches after abortion in South Africa.

Campylobacter jejuni was isolated in pure culture from the vaginal discharge from three German Shepherd bitches after late-pregnancy abortions. The main clinical sign occurring in the bitches was a profuse and odourless haemorrhagic vaginal discharge.

Salmonella isolated from feeds and feed ingredients during the period 1982-1988: animal and public health implications.

The prevalence of Salmonella in southern Africa in farm feeds and by-products of animal origin during 1982-1988 was determined. Salmonella occurred in 5.18% of the farm feed samples and in 9.54% of the by-product samples. Different serovars were isolated, some only once. The findings underestimate the true prevalence of Salmonella in farm feeds and by-products, and is representative of only the most severely contaminated products. The epidemiology of salmonellosis is discussed with special reference to the importance of multiple resistance to antibiotics, the increase in the number of cases of salmonellosis worldwide and "Salmonella free" feeds and foods. More detailed research on the role of farm feeds in the epidemiology of salmonellosis is required. Efforts should be made to increase awareness of the problem, to improve quality management at farm feed production plants and to develop efficient systems to monitor the hygienic safety of feeds and foods.

The biochemical differentiation between Salmonella and Citrobacter.

A number of bacterial isolates which could not be identified as either Salmonella or Citrobacter by conventional biochemical tests and could not be typed as Salmonella with available antisera, were further examined biochemically and by lysis with phage Felix 0.1. Glycerol-positive salmonellae and lysine-positive citrobacters were encountered, which could be confused with the other genus, but when the reactions of such strains were examined in the other tests, accurate identifications could be done. Of the tests examined, glycerol fermentation, the beta-galactosidase test, lysine decarboxylation, sorbose fermentation, galacturonate fermentation and lysis by the phage could be used in the differentiation. These tests in combination, rather than 1 or 2 single tests gave reliable and conclusive differentiation.

The first isolations of Campylobacter mucosalis from pigs in South Africa.

The first isolations of Campylobacter mucosalis in South Africa are described. Isolations were made from a 6-week-old weaner pig with necrotic enteritis and from 2 gingival swabs of suckling piglets from herds with histories of porcine intestinal adenomatosis. The isolates were serologically identified as being serotype A strains.

The isolation of Campylobacter hyointestinalis from a pig in South Africa.

The isolation of a strain of Campylobacter hyointestinalis from a piglet is described. The animal originated from a farm where another animal showed signs of intestinal adenomatosis. The animal from which the isolation was made had diarrhoea, and an enteropathogenic Escherichia coli was also isolated. No pathological changes indicative of intestinal adenomatosis were detected in this animal.

The demonstration of the K-antigen of Campylobacter fetus venerealis using a microtitre agglutination test.

Using specific antisera prepared against heat-treated and untreated cells in a microtitre system, 7 strains of Campylobacter fetus venerealis were examined for the presence of K-antigen. The technique was effective for distinguishing between strains and it was found that 4 out of 7 isolates contained microcapsules, therefore being potentially suitable for vaccine production.

A comparative study of the growth of Campylobacter fetus strains in liquid media.

The growth of C. fetus fetus, C. fetus venerealis and C. fetus venerealis biotype intermedius was examined in 10 liquid media. From the data obtained, a 10% inoculum size and an oxygen level of 6% seemed imperative for consistent growth, especially for the C. fetus venerealis strain. A lowered redox potential obtained by the addition of 0, 1% cysteine-HC1 to the media was stimulatory. The medium which yielded the best growth was the one described by Dennis & Jones (1959). The fastidious C. fetus venerealis strain yielded maximum values of 0,5% packed cell volumes after 48 h cultivation in a microaerophilic atmosphere on this medium. The other strains yielded higher values.