The influence of Mannheimia haemolytica A1 seed culture inoculum cell density on the production of leukotoxin in submerged culture supernatant.

Mannheimia haemolytica leukotoxin is produced during the logarithmic growth phase in submerged culture in RPMI 1640 medium with and without the addition of foetal calf serum or albumin. In order to establish a pattern of optimal leukotoxin production in small volumes in submerged cultures and to define some parameters involved, two high leukotoxin producing Mannheimia haemolytica strains were grown in RPMI 1640 medium containing either FCS or BSA. The cell growth and leukotoxin production abilities of each strain were determined concomitantly every hour in RPMI 1640 medium containing each of the additives over a time period of 6 h. The growth performance of three dilutions of a standardized seed culture inoculum prepared with each of the cultures and additives were simultaneously compared with each other using the above parameters. The different seed culture inoculum dilutions had a definite effect on the time and quantity of leukotoxin production. Both strains demonstrated peak leukotoxin production after 4 h of active growth. The addition of albumin to both isolates gave slightly increased leukotoxin levels, and both showed that the peak leukotoxin was not associated with peak cell concentration. Obvious quantitative differences in the ability of different M. haemolytica strains to produce leukotoxin were noted. Strain 12296 produced optimal leukotoxin concentration from the medium (1/25) dilution of the seed culture inoculum after 4 h, whereas strain 1/10 produced the same concentration with the low (1/5) dilution seed culture inoculum, possibly reflecting the superior production ability of the first strain. However, each strain of M. haemolytica appeared to have its own specific logarithmic cell growth and leukotoxin production pattern. The peak cell density of M. haemolytica grown in submerged RPMI 1640 culture medium cannot be used as an indication of optimal leukotoxin levels.

The production and evaluation of Pasteurella haemolytica leukotoxin in the supernatant of submerged cultures in fermenters.

The optimal production of P. haemolytica leukotoxin in the culture supernatant of a fluid medium is dependent on a number of factors. The leukotoxin has to be produced by using a strain that is known for its ability to produce high quantities of leukotoxin, inoculated into the most suitable type of medium at the correct culture density containing the necessary supplements and harvested after a certain growth period. The volume in which it is produced may also have an influence. Two different procedures are described to produce the leukotoxin in 5 to 15-l quantities in RPMI 1640 medium. The first method used to produce leukotoxin is one that has been repeatedly described since the presence of the leukotoxin was first established in 1978. Using this method seven batches of leukotoxin were produced in litre quantities with leukotoxin activity ranging from 23-67 u/ml. The seed culture inoculum is prepared in brain heart infusion broth, which is centrifuged before the organisms are inoculated into RPMI 1640 medium containing 3.5% foetal calf serum and incubated for only 1 h in a fermenter, after, which the leukotoxin is harvested. An improved alternative method was devised which yielded higher levels of leukotoxin activity by utilising the ability of the P. haemolytica organisms to grow and produce leukotoxin during the logarithmic growth phase in a fermenter. A seed culture harvested in the log phase was prepared in brain heart infusion broth by means of a series of cultures and inoculated into RPMI 1640 containing 3.5% foetal calf serum. Three hours of active growth were allowed during which the leukotoxin was measured by its biological activity and an ELISA assay, and the increase in cell mass by means of the optical density every 30 min. The average leukotoxin biological activity measured 260 u/ml and by means of the ELISA test the leukotoxin concentration measured 315 u/l which is a substantial increase in leukotoxin production. In comparison the average optical density only measured 0.469 at 650 nm. Previous findings were substantiated that the highest cell density was not reflected in the highest leukotoxin activity. It is possible to induce high levels of leukotoxin secretion in submerged cultures with RPMI 1640 medium containing foetal calf serum in the controlled environment of a fermenter in large enough quantities for use as a vaccine by the improved preparation of the seed culture inoculum.

The humoral immune response in cattle after immunization with a multivalent IBR/PI3/Pasteurella haemolytica A1 leukotoxin vaccine.

A multivalent vaccine consisting of inactivated bovine herpes virus-1 (BHV-1), also known as infectious bovine rhinotracheitis virus (IBR), para-influenza type-3 virus (PI3) and the leukotoxin of Pasteurella haemolytica A1, were combined with the addition of aluminium hydroxide as adjuvant, and administered to post-weaned calves, and the serum tested for seroconversion to each antigen. Two groups of calves (n = 150 and n = 68) were used and were randomly divided into three subgroups. The first group of 150 calves were immunized with the multivalent vaccine (three batches) to test its first-stage stability (vaccine 3 months old) and the second group were immunized with the same vaccine 1 year later, in order to test its immunogenicity. A significant increase in titres occurred after the day 0 and 28 immunizations, for each of the three antigens in the multivalent vaccine, as measured on days 0 and 42. Immunoconversion occurred after immunization with the 3-month- and 1-year-old vaccine. The immunogenic stability of antigens in the vaccine was demonstrated after a 1-year period when the vaccine was kept at 4-6 degrees C.

The distribution of Pasteurella haemolytica serotypes among cattle, sheep, and goats in South Africa and their association with diseases.

Over an 8-year period (September 1986 to March 1994), a total of 497 organ specimens from sheep and goats and 96 from cattle, were received for their isolation of Pasteurella haemolytica. They were collected in seven geographical areas in South Africa (as it existed before the April 1994 elections). These areas include the eastern Cape, Transvaal (new name: Gauteng), Nambia, Orange Free State (new name: Free State), Natal (new name: KwaZulu-Natal), western Cape and the northern Cape. This investigation does not represent the statistical incidence of the organism from each region, only the distribution of serotypes isolated from organ specimens submitted from diseased animals in these regions. Pasteurella haemolytica serotype 6 was the most prevalent type isolated from sheep and goats, but was followed closely by types 9 and 2. From cattle, P. haemolytica serotype 1 comprised 39% of the isolates. In sheep and goats, the majority of serotypes were associated with pneumonia, followed by gangrenous mastitis ("blue udder") and septicaemia. The situation in cattle was similar regarding the incidence of pneumonia followed by septicaemia. Up to 33% of the isolates from cattle and sheep specimens were non-typeable.

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.

The antibiotic sensitivity patterns of Bacillus anthracis isolated from the Kruger National Park.

Forty-four isolates of Bacillus anthracis made from carcasses and soil in different localities of an endemic anthrax area in the Kruger National Park, South Africa, were tested by standard disc diffusion for their susceptibility to 18 different antibiotics. These were ampicillin, penicillin G, sulphatriad, streptomycin, clindamycin, gentamicin, fusidic acid, trimethoprim, sulphamethoxazole, chloramphenicol, erythromycin, methicillin, tetracycline (2 different concentrations), novobiocin, cefotaxime, netilmicin, cefamandole and cefoxitin. All the isolates were susceptible to ampicillin, streptomycin, chloramphenicol, erythromycin, tetracycline, methicillin and netilmicin. More than 90% of the isolates were sensitive to clindamycin, gentamicin and cefoxitin, whereas only 84.1% of the isolates were sensitive to penicillin G, 86.4% to novobiocin and 68.18% to cefamandole. Complete resistance in 100% of the isolates was encountered with trimethoprim and sulphamethoxazole, with 95.45% for sulphatriad. Moderate sensitivity occurred with penicillin G (15.9% of the isolates), clindamycin (6.8%), novobiocin (13.6%), fusidic acid (84.1%), cefotaxime (100%), cefamandole (31.8%) and cefoxitin (6.8%). The relevance of the findings to the therapeutic uses of different types of antibiotic in human clinical cases referred to in the literature is discussed.

The biochemical, morphological and virulence profiles of Bacillus anthracis isolated in the Kruger National Park.

The biochemical, morphological and virulence profiles of 44 Bacillus anthracis isolates, obtained from various localities and carcass remains of wild animals in the Kruger National Park, South Africa, were examined. The morphological characteristics tested for included: the formation of capsules on bicarbonate agar, the motility of the vegetative organism, the presence of haemolysis on blood tryptose agar, the sensitivity of the vegetative organism to bacteriophage, the production of lecithinase on egg yolk agar, the liquefaction (hydrolysis) of gelatine and the capability of each isolate to produce mucoid colonies when grown on bicarbonate agar with horse serum in an atmosphere containing CO2. The API 50CHB and 20E systems were used to evaluate the biochemical activity of each isolate. The virulence of each isolate was determined by its LD50, using an inbred line of Balb/C mice. A clear pattern in the biochemical reactions emerged that appeared to be specific for each isolate. On the API 50CHB test strip, only 2 of the 44 isolates gave a 100% positive reaction to all 10 of the biochemical substances to which it was supposed to react, 9 gave positive results to 90%, 19 were positive to 80%, and 14 were positive to 70%. The reactions on the API 20E were completely different from what was expected, with only 1 of the biochemical activities (gelatinase production) showing a positive reaction to all the isolates. The virulence test indicated that 27/44 isolates could be regarded as highly virulent with a LD50 of less than 1,000 organisms, and the rest of the isolates as virulent with a LD 50 of 1,001-10,000 organisms.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of passive immunization on active immunity against Clostridium perfringens type D in lambs.

Lambs in different stages of development of active immunity against Clostridium perfringens type D were treated with partially purified immunoglobulin in an attempt to superimpose a passive immunity on an existing or developing active immunity. Three different studies were undertaken to determine the impact of partial purified immunoglobulins on these vaccinated animals. In 2 of the 3 studies, active immunity was induced by administering the normal routine enterotoxaemia vaccinations and allowing the basic immunity to become established, for a period ranging from 2 weeks for the animals in study 1 and 4 months for those in study 2, before passive immunization with the partially purified immunoglobulins took place. An increase in the epsilon antibody titre occurred in each of the 2 studies after the animals were passively immunized with immunoglobulin, though this increase was not statistically significant (P greater than 0.05). In the 3rd study, when the animals were given the initial vaccination of the Onderstepoort enterotoxaemia oil adjuvant vaccine together with the immunoglobulin, an immediate increase in the epsilon antitoxin titre occurred that was statistically significant (P less than 0.05) 2-14 days after administration. No negative effects were noted on the development of an initial active immunity or an existing active immunity against Clostridium perfringens type D when they were passively immunized with partially purified immunoglobulin.

The passive protection of lambs against Clostridium perfringens type D with semi-purified hyperimmune serum.

Weaned lambs, having a detectable level of maternal antibodies (1-2 units/ml) against C. perfringens type D, showed protective antitoxin levels lasting for 29 days after receiving a single parenteral dose of 200 units/kg hyperimmune serum. Lambs, having no maternal antibodies (less than 0,07 units/ml) to C. perfringens type D but receiving the same dose of hyperimmune serum, maintained protective antibody levels for only 21 days. Three weeks after the titres fell below the minimum protective level of 0,15 units/ml, both these groups were treated again in the same manner. The passive immunity conferred in both groups now lasted for 42 days. When the hyperimmune serum was administered to lambs already immunized by vaccination, a slight increase was noted in the antibody titre.

Purification of the alpha toxin of Clostridium perfringens type A by ultrafiltration and gel chromatography.

Clostridium perfringens type A toxin produced in Jayko & Lichstein medium was subjected to various concentration and purification procedures. The results obtained with 3 different ultrafiltration membranes followed by gel filtration showed that by using Millipore PSED OHV10 and Amicon XM-100 filter membranes in combination, a three-hundred-and-fivefold purification could be achieved as against a twelvefold increase obtained with ammonium sulphate/acetone precipitation. The lecitovitelin test was more sensitive than the haemolytic activity in determining the alpha toxin activity. The optical density, measured at 280 nm, did not reveal any alpha toxin activity in the relevant toxic fractions.

Evaluation of the Venom Ex apparatus in the initial treatment of puff adder envenomation. A study in rabbits.

The Venom Ex apparatus has been evaluated for the treatment of puff adder bite. Rabbits were injected with double the lethal dose of puff adder venom, followed by treatment with the Venom Ex cutting and suction apparatus. Controls received no treatment. The percentage of venom extracted as determined by radial immunodiffusion was very low after intramuscular injection and significantly higher after subcutaneous injection. However, all treated and control animals injected subcutaneously, recovered while all animals injected intramuscularly died, irrespective of treatment. Blood venom levels were extremely low in all animals. Venom Ex treatment did not improve survival or affect local necrosis significantly.

Evaluation of the Venom Ex apparatus in the treatment of Egyptian cobra envenomation. A study in rabbits.

The Venom Ex cutting and suction apparatus for the initial treatment of snakebite was evaluated. Rabbits were injected with radioactive Egyptian cobra venom, and treatment with the Venom Ex followed. The fluid obtained by suction was analysed. All 8 control animals died within 4 hours; Venom Ex treatment resulted in the recovery of 7 out of 8 rabbits, after double the lethal dose of venom, providing treatment was started early. However, if treatment was delayed or if the dose of venom was high, there was a marked increase in the mortality. The amount of venom extracted was insufficient to account for the recovery of the animals. In one group of rabbits trauma was applied to the injection site without lacerating the skin and without removal of venom. About half of these animals recovered. However, this was less efficient than the Venom Ex treatment. Trauma apparently retards absorption of venom and increases survival. The possible reasons for this novel finding are discussed.

Mycobacterium tuberculosis in a closed colony of baboons (Papio ursinus).

An outbreak of tuberculosis in a nonhuman primate colony involved 11 of 91 (12.1%) baboons (Papio ursinus). Tuberculin tests identified 80%, X-ray screening 10% and throat swab bacteriology 30% of diseased animals. Cough was a misleading indicator of tuberculosis. Stress resulting from experimental interference with animals was unrelated to disease development. Twice as many females as males were attacked. Screening and prophylactic, therapeutic and preventive measures are discussed.

Postoperative Clostridium septicum dermatitis simulating toxic epidermal necrolysis.

Widespread detachment of the epidermis as a result of Clostridium septicum infection after a laparotomy for intestinal obstruction is described. A clinicopathological picture, hitherto undescribed, is outlined.