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.

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.

Chlamydial infection in asymptomatic infertile men attending an andrology clinic.

Urethral swabs, first-catch urine or urine collected at least 2 h after the previous micturition, and semen swabs were collected from 131 asymptomatic men during the routine workup for infertility at the andrology clinic at Ga-Rankuwa Hospital, Medunsa. The urethral and seminal swabs were used for enzyme immunoassay (EIA) (IDEIA III), tissue culture, and direct immunofluorescent antibody (DFA) test (IMAGEN) to detect Chlamydia trachomatis, and similarly the urine was tested by EIA. In 82/131 (62.6%) cases all tests for chlamydiae were negative and in 49/131 (37.4%) cases at least one test was positive. Tissue culture detected 24/131 (18.3%) as positive for C. trachomatis. Urethral swab EIA detected 33/131 (25.2%) and DFA 34/131 (26%) positive patients. Urine EIA was positive in 33/131 cases (25.2%). Semen EIA was positive in 35/131 (26.7%) of cases of whom 7/131 (5.3%) were positive in semen EIA only (all samples were conformed by PCR). It would seem, therefore, that testing for the presence of chlamydia was incomplete if semen samples were not included. The positivity in only semen samples raises the possibility that the organisms are harbored in the epididymis, seminal vesicles, or the prostate.

Comparison of urine with urethral swabs for the detection of Chlamydia trachomatis in men attending an STD clinic.

Urethral swabs and first-catch urine specimens for the detection of Chlamydia trachomatis were collected from 370 black men with urethritis who attended a sexually transmitted disease (STD) clinic in Pretoria. An enzyme immunoassay (EIA) for C. trachomatis was carried out on all urethral swabs and urine specimens. Chlamydial culture and a direct immunofluorescent antibody (DFA) test (Imagen, Dako, UK) were also carried out on urethral swabs; DFA was used for confirmation of urine EIA positives. Based on culture and/or DFA, C. trachomatis was detected in 96 (26%) urethral swab specimens. The sensitivity of urine EIA investigation was 94% and the specificity 99%, compared with those of urethral swab EIA which were 97% and 99% respectively. The positive and negative predictive values for urine were 96% and 98% compared with 96% and 99% respectively for urethral swabs. Urine examination was therefore sensitive and specific compared with urethral swab examination in these STD patients. In view of this, the advantage of urine as an alternative to urethral swabs for C. trachomatis detection is that sampling is non-invasive and non-traumatic.

The microbial aetiology of summer paediatric gastroenteritis at Ga-Rankuwa Hospital in South Africa.

Stool material from seventy-eight children below the age of three years was examined for the presence of various enteropathogens. The patients had been admitted to the Ga-Rankuwa hospital for rehydration therapy. A causative agent was identified in 76.9% of the cases studied. The most prevalent organisms identified were 38.5% entero-toxigenic Escherichia coli (ETEC), 25.6% Cryptosporidium parvum, 15.4% Campylobacter sp., 14.1% enteric adenoviruses and 12.8% rotavirus. In 38.5% of cases, a mixed infection was observed with up to four different organisms being identified from a single patient. ETEC elaborating heat-labile toxin (LT) found together with C. parvum was the most common combination seen in mixed infections.

Incidence of toxigenic Campylobacter strains in South Africa.

Campylobacter strains can produce a heat-labile cytotonic toxin (CTON) and various cytotoxins (CTOX). Of 22 South African Campylobacter strains tested, 86% were toxigenic (77% produced CTON, 41% produced CTOX, and 32% produced both types) and 14% were toxin negative. Campylobacter jejuni strains were 67% CTON positive and 47% CTOX positive, whereas Campylobacter coli strains were 100 and 29% positive, respectively.

Comparison of the effect of antibiotic dry cow teat canal and intramammary dry cow therapy of dairy cows on the prevalence of teat canal and intramammary infections at calving.

The specific therapy of bacterial colonization of the teat canals of dried-off dairy cows by means of small amounts (33 mg/0.25 ml and 14 mg/0.1 ml) of a procain benzyl penicillin-dihydrostreptomycin sulphate combination has been investigated. Of 36 teat canals treated with 0.25 ml antibiotic preparation each, 24 (66.6%) were infected at the beginning of the dry period, whereas at its termination only 7 (19.4%) showed bacteriologically positive swab cultures. By treating a further 43 quarters with 0.1 ml quantities of an antibiotic preparation each, the prevalence of infection dropped from 32.5% to 14%. Untreated controls (20 quarters) showed no significant change. Cultures of teat canal swabs were generally twice as sensitive for indicating the presence of bacteria compared to foremilk sample cultures. Staphylococcus aureus was the most commonly isolated organism, followed by Streptococcus agalactiae, Corynebacterium bovis and Staphylococcus epidermidis.

Simultaneous isolation of anaerobic bacteria from udder abscesses and mastitic milk in lactating dairy cows.

A variety of non-sporulating anaerobic bacterial species were isolated from udder abscesses in 10 lactating dairy cows. Fifty percent of the abscesses yielded multiple anaerobic species and the other 50% only 1 species. The anaerobic bacteria, however, were always accompanied by classical facultative anaerobic mastitogenic bacteria. In four of the five cows also afflicted with mastitis in the quarters with abscesses, the anaerobic and facultative anaerobic bacteria were identical. Peptococcus indolicus was the most commonly isolated organism followed by Eubacterium and Bacteroides spp. Bacteroides fragilis was resistant to penicillin, ampicillin and tetracycline.

The effect of lincomycin-neomycin treatment on experimental anaerobic bacterial bovine mastitis.

Three healthy lactating quarters of a Friesland cow were each experimentally infected with a pure culture of a strain of either Bacteroides fragilis, Eubacterium lentum or a Peptostreptococcus sp. respectively. The onset and progression to clinical mastitis was monitored 12 hourly by examination for clinical signs of inflammation, bacterial culture, somatic cell counts and with a strip cup. All infected quarters developed clinical mastitis within 24 hours. The 2 quarters infected with B. fragilis and E. lentum respectively were treated 4 times consecutively at 12 hour intervals, commencing at 24 h by intramammary instillation of 10 ml of a mixture containing 200 mg lincomycin hydrochloride, 200 mg neomycin sulphate and 5 mg methylprednisolone (Lincocin Forte, Upjohn). Both quarters became clinically normal and no bacteria could be detected in the secretions 12 hours after the first treatment. At 36 hours the strip cup became negative, and the somatic cell count dropped to less than 500 X 10(3) at 72 hours after the initial treatment. The quarter infected with a Peptostreptococcus sp. was unable to overcome the infection by natural means when intramammary treatment was delayed for the first 36 hours after the onset of clinical mastitis. Subsequent treatment of this quarter gave results similar to those treated earlier.

The frequency and some characteristics of anaerobic bacteria isolated from various forms of bovine mastitis.

The prevalence of strictly anaerobic bacteria in the secretions from untreated cases of mastitis in lactating dairy cows was investigated. The study involved 147 Friesland cows in 12 highveld herds. All herds yielded cows with anaerobic udder infections. No anaerobic bacteria were recovered from cows with normal quarters or those with latent aerobic infections. Only anaerobes were present in 10% of so-called 'aseptic' mastitis cases. A variety of anaerobic organisms was isolated concurrently with facultative bacteria from 5,3% and 58,8% of cases classified as subclinical and clinical respectively. Peptococcus spp. was associated with Corynebacterium pyogenes and Bacteroides spp. with Staphylococcus aureus and/or Streptococcus agalactiae in 80% anaerobic udder infections. Gram positive anaerobic species were mostly sensitive to penicillin-G but all the Gram negative rods were resistant. In addition, all B. fragilis strains produced beta-lactamase. The ability to produce heparinase was demonstrated in one strain of Peptococcus indolicus and a Peptostreptococcus sp.

Isolation and significance of anaerobic bacteria isolated from cases of bovine mastitis.

The role of obligate anaerobic bacteria in the aetiology of mastitis of lactating dairy cows was investigated. Anaerobes were isolated from 12% of lactating mastitic cows, which were representative of 50% of the 10 dairy herds examined. Bacteroides fragilis was the most frequently isolated organism of (50%), followed by Peptococcus indolicus (33%), Eubacterium lentum (33%), E. aerofaciens (17%), Propionibacterium granulosum (17%) and an anaerobic Streptococcus sp. (17%). These obligate anaerobes were always isolated together with organisms classically involved in mastitis. It was possible to induce overt clinical mastitis in healthy lactating udders within 24 hours by infection with single pure cultures of anaerobes via the teat canal. All B. fragilis strains were resistant to penicillin G and tetracycline. In addition, one strain was also resistant to ampicillin, cephalothin and amoxicillin. Anaerobic gram positive cocci and bacilli were sensitive to most antibiotics. These findings imply an important role for anaerobes in the aetiology of mastitis.

A microbiological investigation of acute summer gastroenteritis in black South African infants.

A microbiological investigation of Black infants suffering from severe acute summer gastroenteritis revealed enteropathogenic agents in 30 out of 37 patients (81%). Enterotoxigenic bacteria were isolated from 15 patients (41%). A total of 16 enterotoxigenic strains were isolated, comprising 9 enterotoxigenic Escherichia coli strains secreting labile and stable toxin on their own and in combination, and labile-toxin secreting strains of Klebsiella pneumoniae (4), Enterobacter cloacae (2) and Proteus vulgaris (1). In the case of the latter three species, however, 6 out of the 7 strains were isolated from patients who were excreting other enteric pathogens, whereas only 2 out of 9 enterotoxigenic E. coli patients had concomitant infections with other pathogens. No invasive bacteria were isolated except for 2 shigella strains. Salmonella and shigella strains were found in four patients. No correlation was found between the enteropathogenicity of E. coli and its serotype. Rotavirus was observed by negative staining electron microscopy in only two patients (6%) but using a reverse complement fixation test rotavirus antigen was detected in the stool of 17 out of 35 patients (49%). The low EM detection rate may well be due to the patients being admitted for treatment late in the course of their illness when the degree of viral shedding has decreased below EM detectability. No significant difference in clinical presentation was noted between the various aetiological agents. Only one patient was being solely breast-fed compared to 16% of control non-diarrhoeic infants. Evidence of malnutrition was noted in over half of our patients.