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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Immune mechanisms in C57B1 mice genetically resistant to Trypanosoma congolense infection. II. Aspects of the humoral response.

Some aspects of the humoral response in trypanotolerant C57B1 mice and susceptible A/J mice were investigated to determine the possible basis of trypanotolerance. When the hepatic uptake of 75Se-labelled T. congolense by infected mice was measured as an index of antibody production, it was found that only C57B1 mice could remove circulating labelled parasites, this ability persisting for several weeks after infection. Estimation of the immunoglobulin concentrations in both strains of mice showed that C57B1 mice developed a pronounced IgM response during the first parasitaemic wave, while A/J mice did not. Over the same period the IgM concentrations in C57B1 mice initially fell, but recovered at the time of peak parasitaemia. In contrast, A/J mice showed a continual fall in total IgG concentrations in the circulation until death 10 days after infection. Finally, it was shown that during the initial rising parasitaemia, the plaque forming cell responses of both strains of mice to sheep red blood cells were normal indicating that neither strain of mice was immunosuppressed. Also, A/J mice vaccinated with irradiated T. brucei on day 4 and C57B1 mice vaccinated either on day 4 or day 60 of a T. congolense infection were able to mount an effective immune response to the vaccine, as judged by the hepatic uptake of radiolabelled parasites. All of the results indicate that the trypanotolerance of C57B1 mice depends, at least in part, on their more efficient antibody response.

Regulation of the growth and differentiation of Trypanosoma (Trypanozoon) brucei brucei in resistant (C57Bl/6) and susceptible (C3H/He) mice.

While Trypanosoma brucei brucei GUTat 3 were equally infective for C3H/He and for C57Bl/6 mice at doses ranging from 5 to 5 x 10(3) organisms and had similar prepatent periods in both strains of mice, infected C57Bl/6 mice displayed lower parasitaemia, shorter times to parasite wave remission and survived for a longer time than infected C3H/He mice. Parasite growth and differentiation rates and host immune responses were similar for the first 5 days in both strains of mice after infection with 10(3) T.b.brucei GUTat 3 but, thereafter, parasite differentiation proceeded more rapidly and specific antibodies reached higher titres in C57Bl/6 than in C3H/He mice. In contrast, parasite growth and differentiation rates were similar in irradiated mice of both strains. Furthermore, following inoculation of intact mice with irradiated T.b.brucei GUTat 3, C3H/He mice actually mounted higher titred antibody responses than C57Bl/6 mice showing that they were not intrinsically defective in their capacity to respond to GUTat 3 antigens. Parasite differentiation occurred at the same rate in irradiated (650r) C57Bl/6 mice and in irradiated C57Bl/6 mice reconstituted with syngeneic spleen cells although T.b.brucei GUTat 3 specific antibody was detected in the latter mice prior to peak parasitaemia. Furthermore, it was shown directly in C57Bl/6 mice that there was no selective destruction of slender form T.b.brucei GUTat 3 parasites during the phase of accumulation of stumpy form parasites. These studies indicate that the more rapid differentiation of T.b.brucei GUTat 3 parasites in infected C57Bl/6 mice as compared to infected C3H/He mice was unlikely to be directly related to the more efficient antibody response in the infected C57Bl/6 mice. The observations suggest that there might be an association between host mechanisms which regulate differentiation of T.b.brucei parasites and those which regulate antibody responses.

Immune mechanisms in C57Bl mice genetically resistant to Trypanosoma congolense infection. I. Effects of immune modulation.

The effect of immune modulation on the pattern and course of infection with T. congolense was investigated in a strain of mice (C57Bl) which is known to possess a significant degree of trypanotolerance, and a susceptible strain (CFLP) which rapidly succumbs to infection. Immunosuppression of C57Bl mice by splenectomy, cyclophosphamide treatment or gamma irradiation reduced their survival to near that of susceptible strains of mice. In contrast, attempts to enhance the immune response of susceptible CFLP mice using either a variety of immunostimulants, simultaneous vaccination with irradiated parasites at the time of infection, passive immunization or reducing the number of parasites used for infection, failed to confer a level of protection comparable to that of C57Bl mice. It was concluded that the basis of trypanotolerance, although immunological in nature, is associated with, as yet, undetermined factors.

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.

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.