Immunogenicity and molecular characterisation of staphylococcal delta haemolysin.

In rabbit antisera to staphylococcal delta haemolysin the haemolysin-neutralising activity was associated with the IgG fraction as well as with the alpha and beta lipoproteins. On an equal-weight basis, the neutralising capacity of specific antibody was not significantly greater than that of serum alpha or beta lipoproteins. Anti-alpha haemolysin was not detected in any of the anti-delta-haemolysin antisera. Amino-acid analysis of delta haemolysin showed that lysine, aspartic acid, threonine and isoleucine were the predominant amino acids present; histidine, arginine, proline, cysteine and tyrosine were absent. The molecular weight of delta-haemolysin in phosphate-buffered saline was approximately 210 000.

Immunological clearance of 75Se-labelled Trypanosoma brucei in goats.

An experiment was conducted to determine, under different conditions, the capacity of young adult East African goats to eliminate intravenously inoculated [75Se]selenomethionine-labelled Trypanosoma brucei from the bloodstream. Over 80% of labelled trypanosomes, preincubated for 1 h in inactivated normal goat serum, were detectable in the circulation 1 h after inoculation into normal goats. By contrast, after incubation in serum from goats which had been immunised against the homologous trypanosome clone, parasites were largely removed from the bloodstream within 5 min after inoculation. When the goats were necropsied 1 h after the inoculation of radiolabelled trypanosomes, 50% of the injected activity was found in the liver and lungs, the contribution of each organ being dependent to some extent on whether the inoculum was via a mesenteric or the jugular vein. The same result was obtained when labelled parasites were incubated in normal goat serum, and then inoculated into immunised goats; thus, rapid blood clearance occurred, and high activity was detected in the lungs and liver. The results confirm those of previous studies in laboratory mice in which the removal of trypanosomes from the circulation of an immune animal was achieved primarily by uptake of opsonised trypanosomes by elements of the mononuclear phagocytic system.

Haemorrhagic lesions resulting from Trypanosoma vivax infection in Ayrshire cattle.

Infection of Ayrshire cattle with a stock of Trypanosoma vivax from the Galana Ranch, Kenya, resulted in an acute disease characterised by profound anaemia and haemorrhage, which reached maximum severity between 3 and 5 weeks after infection. Bleeding from the ears, nose and rectum occurred. At necropsy, petechial and ecchymotic haemorrhages were widespread, but were particularly severe in the gastrointestinal tract. In confirmation of the gross findings, congestion, haemorrhage and degenerative changes in most tissues and organs were found histologically. Thrombi were seen in the lymphatic vessels and clots of fibrin were present in the ventricles of the brain. The anaemia was a consequence of frank blood loss through haemorrhaging, exacerbated by erythrophagocytosis of deformed red blood cells, whose occurrence was indicative of microangiopathic changes. Animals were euthanised between 23 and 36 days after infection when they became recumbent with PCV values as low as 9%. There is no doubt that animals affected by this syndrome in the field would die within a few weeks of infection, if left untreated.

Factors influencing the duration of isometamidium chloride (Samorin) prophylaxis against experimental challenge with metacyclic forms of Trypanosoma congolense.

The duration of a single isometamidium chloride (Samorin) prophylactic treatment against Trypanosoma congolense ILNat. 3.1 and T. congolense IL 285 was examined in 24 Boran steers with regard to (1) the dose of drug, (2) the level of metacyclic challenge and (3) the influence of infection with an unrelated serodeme at the time of treatment. The cattle were repeatedly challenged at monthly intervals between 2 and 7 months following treatment, either by five infected Glossina morsitans centralis or by intradermal inoculation of 5 X 10(3) or 5 X 10(5) in vitro-derived metacyclic trypanosomes. A dose of 1 mg kg-1 afforded complete protection for 4 months and 0.5 mg kg-1 for 3 months against the two T. congolense serodemes examined, irrespective of the method or weight of challenge. In another group of cattle, which had an established infection at the time of treatment, the duration of chemoprophylaxis against an unrelated serodeme was the same as the other groups which had no previous experience of trypanosome infection. Antibodies to metacyclics did not appear in any of the cattle as long as the chemoprophylaxis was effective. An exception to this was the group challenged with 5 X 10(5) in vitro-derived metacyclic parasites, in which low antibody titres were detected. In all cases these proved to be non-protective. It was concluded that, under the experimental conditions employed, (1) there was a direct relationship between drug dosage and the duration of chemoprophylaxis, (2) the weight of metacyclic challenge did not affect the duration of chemoprophylaxis and (3) when used to treat an existing infection, isometamidium chloride exerted the same degree of chemoprophylactic activity.

Therapeutic and prophylactic activity of isometamidium chloride in Boran cattle against Trypanosoma vivax transmitted by Glossina morsitans centralis.

Ten Boran steers were infected with Trypanosoma vivax, transmitted by Glossina morsitans centralis; five steers with a T vivax clone from Nigeria and five with a T vivax clone from Kenya. Eleven days after infection all 10 animals were treated with 0.5 mg kg-1 isometamidium chloride. Four steers infected with the Nigerian T vivax and all five infected with the Kenyan T vivax were completely cured. When different steers received a single prophylactic dose of 0.5 mg kg-1 isometamidium chloride and subjected to monthly tsetse-transmitted challenge with the same T vivax clones, complete protection was afforded for at least two months against challenge with the Nigerian T vivax, but for less than one month against the Kenyan T vivax. The findings indicate that the level of sensitivity of a T vivax population to the prophylactic activity of isometamidium chloride cannot be concluded from sensitivity studies based on the therapeutic action of the drug.

The susceptibility of strains of mice to infection with Trypanosoma congolense.

The survival times of a number of strains of mice after infection with a stabilate of Trypanosoma brucei or of T congolense were examined. The mean survival times of all the strains when infected with T brucei TREU 667 ranged from 27 to 63 days. Greater disparity was observed after infection with T congolense GVR1 where the mean survival time ranged from seven (BALB/c) to 74 days (C57 Bl). It is suggested that the C57 Bl mouse might provide a laboratory model for the study of trypanotolerance in cattle.

Development of an enzyme-linked immunosorbent assay for the detection and measurement of the trypanocidal drug isometamidium chloride in cattle.

An enzyme-linked immunosorbent assay (ELISA) was developed to measure accurately levels of the trypanocidal drug isometamidium in the serum of treated cattle. The assay requires only 5 microliters of test serum, is sensitive to a level of 0.5 pg ml-1 and is highly specific. Cross reactivity does not occur with the two other widely used trypanocidal drugs diminazene aceturate and homidium bromide. Serum drug levels are detectable for up to six months in cattle after a single dose of 1 mg kg-1 intramuscularly, the maximum period under field conditions for which effective prophylaxis can be maintained against tsetse challenge. Application of the assay will aid the rationalisation of treatment campaigns and assist in assessing the occurrence of drug-resistant trypanosome populations.

Immunosuppression in bovine trypanosomiasis: studies with louping-ill vaccine.

The antibody response to louping-ill virus vaccine was examined in mice infected with Trypanosoma brucei and T congolense, and in Ethiopian cattle experimentally infected with T brucei, T congolense and T vivax. In mice the antibody response was completely suppressed, while in cattle infected with T congolense and T vivax the antibody response to the vaccine was only 10 per cent that of uninfected animals. In contrast, the response of cattle infected with T brucei was not significantly reduced, and this was attributed to their relatively light and transient parasitaemias. Trypanocidal chemotherapy (diminazine aceturate) administered on the same day as vaccination largely restored the competence of the immune response of both mice and cattle infected with T congolense. The use of such drugs should be considered when cattle are vaccinated in trypanosome endemic areas.

Isometamidium chloride prophylaxis against Trypanosoma congolense challenge and the development of immune responses in Boran cattle.

Twenty-four Boran cattle were injected with isometamidium chloride (1 mg/kg bodyweight) to investigate the duration of drug-induced prophylaxis against infection by metacyclic forms of Trypanosoma congolense and to determine if specific antibody responses to the organism were mounted by animals under chemoprophylactic cover. Complete protection against either single challenge by five tsetse flies infected with T congolense, or repeated challenge at monthly intervals by five tsetse flies, lasted for five months. Six months after treatment, two-thirds of the cattle were resistant to challenge, irrespective of whether subjected to single or multiple challenge with trypanosome-infected tsetse flies, or titrated doses of in vitro-cultured metacyclic forms of T congolense (5 X 10(2) to 5 X 10(5) organisms), inoculated intradermally. No animal which resisted infection developed detectable skin reactions at the site of deposition of metacyclic trypanosomes or produced trypanosome-specific antibodies. It was concluded that drug residues effectively limited trypanosome multiplication at the site of deposition in the skin, thus preventing subsequent parasitaemia or priming of the host's immune response.

Maternally derived immunity in young mice to infection with Trypanosoma brucei and its potentiation by Berenil chemotherapy.

Young mice which were allowed to suckle, from birth, a mother infected with Trypanosoma brucei, or a mother whose infection had been cured before parturition with Berenil chemotherapy, were themselves immune to homologous trypanosome challenge. This immunity extended until approximately 25 days of age, and was transmitted in the colostrum/milk of the mother. Mice born of infected mothers, but transferred at birth to normal foster mothers, were susceptible to trypanosome infection. Drug prophylaxis in normal newborn mice was also effective for approximately 25 days, but in mice which, in addition, received colostral antibody from the mother, combined immunochemoprophylaxis protected the offspring for 40-50 days. Since the combination of protective strategies continued to resist challenge beyond the stage when, on its own, each component's efficacy had decayed, it may be of practical value as an approach to improved disease control under certain field conditions where trypanosomiasis prevails.

Extravascular foci of Trypanosoma vivax in goats: the central nervous system and aqueous humor of the eye as potential sources of relapse infections after chemotherapy.

Relapse of parasitaemia after drug treatment of trypanosome infection is normally attributed to drug-resistance on the part of the parasite, under-dosage of the drug or reinfection of the host. In addition, inaccessibility of parasites to drug through sequestration in privileged extravascular sites has been shown in the past to occur with Trypanosoma brucei, and we have obtained evidence that extravascular foci of T. vivax can also serve as a source of relapsing infections. Infection of goats with a West African stock of T. vivax resulted in severe illness, which was fatal if untreated. During the terminal stage of an acute infection, clinical signs of central nervous system involvement were apparent. Histologically, the choroid plexus was swollen and oedematous, and in some cases meningitis or meningoencephalitis was seen. Trypanosomes could be detected in the cerebrospinal fluid, and also extravascularly in the choroid plexus and meninges. In three cases they were present in the aqueous humor, associated with corneal cloudiness or opacity. Treatment of 2 goats with the trypanocidal drug diminazene aceturate eliminated parasitaemia, but infections in both relapsed about 6 weeks later, despite trypanosomes being undetectable in the bloodstream during the intervening period. We conclude that the relapse infections were caused by reemergence of trypanosomes from the CNS and/or the eye, where sequestered parasites may have been inaccessible to the trypanocide.

Peptidase in the plasma of mice infected with Trypanosoma brucei brucei.

The plasma of mice infected with pleomorphic Trypanosoma brucei brucei contains a peptidase which has the same electrophoretic mobility on starch gels as a parasite peptidase. An enzyme with this electrophoretic mobility was not detected in the plasma of uninfected mice. The molecular weight of this enzyme in either parasite lysate or plasma from infected mice was approximately 40,000 Da when assayed on a size exclusion column using high-performance liquid chromatography. The enzyme can cleave the dipeptides leu-ala, val-leu and pro-leu, but not the dipeptide phe-ala. The enzyme also cleaved the tripeptides tyr-tyr-tyr and leu-gly-gly. Another parasite peptidase which migrates on starch gels to a different position than the above-mentioned peptidase cleaved the dipeptides leu-ala, val-leu and pro-leu but could not cleave the tripeptides tyr-tyr-tyr or leu-gly-gly. Furthermore, incubation of this parasite peptidase with normal mouse plasma at 37 degrees C resulted in an apparent loss of detectable activity. It is postulated that the plasma of mice modifies either the charge or enzymic activity of this peptidase. We speculate that the parasite peptidase present in the plasma of mice infected with T. brucei could contribute to pathogenesis.

Differences in sensitivity of Kenyan Trypanosoma vivax populations to the prophylactic and therapeutic actions of isometamidium chloride in Boran cattle.

Isometamidium chloride was administered as a single prophylactic dose of 0.5 mg kg-1 body weight to each of 10 Boran (Bos indicus) steers. At monthly intervals following drug administration, groups of five cattle each were challenged with one of two different Trypanosoma vivax populations transmitted by infected Glossina morsitans centralis; one with a stock (IL 2982) from Galana, Kenya and the other with a stock (IL 2986) from Likoni, Kenya. Prophylaxis was afforded for less than one month against the Galana T. vivax and for one month against the Likoni T. vivax. In a therapeutic study a further 10 Boran steers were similarly infected with either of the T. vivax populations; five steers per population. Eleven days after infection all animals were treated with 0.5 mg kg-1 isometamidium chloride and all were cured. These findings demonstrate that, as defined in the field, the two Kenyan T. vivax populations express a high level of resistance to the prophylactic action of isometamidium yet a low level of resistance to the therapeutic action of the drug. The results also indicate that differences in drug resistance between different isolates play a major role in determining the apparent period of prophylaxis afforded by isometamidium chloride.

Susceptibility of different breeds of goats in Kenya to experimental infection with Trypanosoma congolense.

To assess whether there was any evidence of genetic resistance to African trypanosomiasis, five breeds of goat (East African, Galla, and crossbreds between East African and Galla, Nubian or Toggenburg) were experimentally infected with Trypanosoma congolense either by needle inoculation or by tsetse-transmission. The goats had not been previously exposed to trypanosomiasis. With both methods of infection all breeds were found to be highly susceptible and suffered severe disease. Following tsetse-transmitted infection no significant differences were observed between breeds in the development, duration and size of the chancre reaction or in the degree and duration of parasitaemia. While Nubian goats developed anaemia more rapidly than the other breeds, all animals experienced a pronounced reduction in packed red cell volume. Similarly following needle inoculation no differences were found between breeds in the severity of anaemia or in the kinetics of parasitaemia. Immune responses against both metacyclic and bloodstream trypanosomes of the infecting serodeme were similar in all breeds as were the erythropoietic responses to the infection. No alterations in leucocyte parameters occurred.

Immunological clearance of 75Se-labelled Trypanosoma brucei in mice. II. Mechanisms in immune animals.

Using trypanosomes labelled with [75Se]-methionine a series of experiments was conducted to investigate the respective roles of antibody, macrophage activtion and complement in the removal of trypanosomes from the circulation of immune mice. It was found that clearance in such animals is largely accomplished by antibody-mediated hepatic phagocytosis, which, at least in passively immunized animals, is dependent on opsonization involving C3. No evidence was found to suggest that intravascular lysis or activated macrophages are involved in immune clearance.

Genetic resistance to Trypanosoma congolense infections in mice.

The mechanisms of genetic resistance or "trypanotolerance" to infection with Trypanosoma congolense were investigated in two strains of mice. One strain C57BL, is outstandingly resistant to most stabilates of T. congolense and can survive for over 80 days, whereas CFLP, in common with most other strains, generally succumbs in less than 20 days. Evaluation of several pathophysiological and immunological parameters showed that after infection both strains initially developed similar levels of parasitemia, anemia, biochemical derangement, and immunosuppression. The most outstanding difference was after day 8 postinfection, when the susceptible strain (CFLP) sustained high levels of parasitemia (10(9) trypanosomes per ml) until death 2 to 4 days later, whereas the resistant strain (C57BL) showed a marked decrease to less than 10(6) trypanosomes per ml by day 10 postinfection. Clear evidence that this was associated with the presence of trypanocidal antibody in the resistant mice was provided by the results of an infectivity neutralization test on serum collected from each strain at 10 days postinfection. Chronically infected C57BL mice showed declining waves of parasitemia and a slow restoration of most hematological and biochemical indexes to near normal levels by 80 days postinfection, although at this stage they remained immunosuppressed.

Immunological clearance of 75Se-labelled Trypanosoma brucei in mice. I. Aspects of the radiolabelling technique.

A reliable and simple technique for the in vivo labelling of Trypanosoma brucei with [75Se]-methionine was developed. Between 97 and 99% of the radioactivity was protein bound in the trypanosomes and spontaneous elution in vitro was less than 10% over 4 h. The fate of the labelled trypanosomes after i.v. injection into normal and immune mice was studied. Whilst the vast majority of parasites remained in the circulation of normal animals they rapidly disappeared from the blood of immune animals. In the latter the liver was found to be the principal site of phagocytosis removing over 50% of the radiolabelled parasites within 15 min of injection.