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African trypanosomiasis caused by tsetse-borne heteroxenous trypanosomes (T. vivax vivax, T. congolense congolense, and T. brucei brucei) is known as Nagana. Formerly the term was restricted to infections caused by T. b. brucei. Today, the term “trypanosomiasis” is also used as a collective word for all animal trypanosomiasis. The severity of disease may depend on several factors, such as trypanosome species, strain variants, infection dose (low or high tsetse risk), and species of host. Infections can vary from acute (T. c. simiae infections in pigs, T. b. evansi infections in camels) to usually mild or almost inapparent (T. b. brucei infections in cattle). In typical cases, African trypanosomiasis is a wasting disease with clinical signs like anemia, leucopenia, thrombocytopenia, plasma biochemical changes and lesions in some tissues and organs. The disease produces slowly progressive loss of condition accompanied by increasing weakness and extreme emaciation, leading eventually to collapse and death. T. v. vivax causes the most important form of trypanosomiasis in cattle in West Africa and elsewhere. The infection may be asymptomatic, subacute, peracute or chronic. Hemorrhagic T. vivax outbreaks have been reported from farmers in Kenya and Uganda with considerable deaths of cattle. Symptoms were anemia, bleeding through the skin and ears (prior to death), petechial hemorrhages on the tongue and enlarged spleen (for more information on hemorrhagic T. vivax see: Use of Drugs in the Field to Control Cattle Trypanosomiasis). T. c. congolense produces the most severe form of animal trypanosomiasis in East and Central Africa. Serious disease and death may occur in cattle, horses, and dogs. T. b. evansi also occurs in a dyskinetoplastic form in Central and South America (synonyms include T. equinum and T. venezuelense) where it is regarded as a separate species. T. brucei equiperdum produces a venereal disease (= Dourine) in equids in Northwest Africa, Ethiopia, Central and South America, the Middle East and Asiatic Russia. The disease is usually transmitted by coitus, and infrequently by biting flies or infective discharge. Apart from the typical salivarian or stercorarian pathway of infection any trypanosome can also be transmitted mechanically (e.g., artificially by “syringe passage”) without undergoing cyclical development in a vector as T. vivax infections of ruminant livestock in South and Central America. Noncyclical transmission can be done in nature by blood-sucking insects, such as Tabanus spp. and Stomoxys spp. flies (Diptera). In South America vampire bats should also be a vector transmitting T. brucei evansi infections in horses. The disease is known as Murrina (Panama) or Derrengadera (Venezuela). T. b. equiperdum infections in horses and donkeys may be transmitted by coitus.
Economic loss due to cattle trypanosomiasis is difficult to assess, but the fact that livestock in Africa are treated with more than 30 million doses of trypanocidal drugs each year may give some indication of the importance of this problem. The impact of disease extends over approximately 9 million km2 of Africa between the southern border of the Sahara in the north and the Limpopo in the south (sub-Saharan Africa), and threatens more than 50–70 million animals in 37 African countries. Partly a result of this disastrous situation is that Africa produces about 70 times less animal protein per unit area than Europe.
There are two groups of tsetse-transmitted organisms, which can be distinguished: (1) the hematic group, including T. c. congolense and T. v. vivax and confined to the blood and lymphatic systems and (2) the humoral group, including T. b. brucei, T. brucei rhodesiense and T. b. gambiense (Trypanocidal Drugs, Man/Drugs Acting on African Trypanosomiasis (Sleeping Sickness) of Humans). In addition to occurring in plasma, species of the humoral group are also present in body cavity fluids and intercellular tissue. Parasites of this group are parasitemic only in the terminal stages of the infection, and the chief pathological changes caused by these trypanosomes are extensive inflammatory, necrotic, and degenerative reactions (tissue damage), probably associated with release of kinins and fibrinogen degradation products. In contrast, parasites of the hematic group produce mainly a severe anemia, which determines the severity of disease. Although the anemias produced by T. v. vivax and T. c. congolense are equally serious, the mechanism of pathogenicity may be different for each species. T. c. congolense can also develop outside the circulatory system. Thus, the different distributions of the trypanosomes in the body of host result in varying susceptibilities to trypanocides depending on their pharmacodynamics (mechanisms of drug action) and pharmacokinetics (disposition and fate of drugs in the body). Relapse of infection, i.e., return of patent parasitemia after its apparent cessation by drug administration, may occur in chronic T. b. brucei infections (Trypanocidal Drugs, Man/Drugs Acting on African Trypanosomiasis (Sleeping Sickness) of Humans: late stage of trypanosomiasis = sleeping sickness of man). The relapse due to the appearance of trypanosome populations from privileged sites, such as the cerebrospinal fluid and/or intercellular tissue spaces (parasites from the latter site may also be the cause of relapse in T. c. congolense infections). Commonly used drugs, such as diminazene, isometamidium and homidium do not have the ability to cross the blood-brain barrier or produce constant trypanocidal concentrations in body cavity fluids and intercellular tissues that kill trypanosomes. Relapse in chronic T. b. brucei infections is evident when chemotherapy was started too late. This is of considerable interest because drug sensitivity changes as the infection progresses. Complete cure is usually achieved when drugs are given in the early stage of infection. In late-stage T. b. brucei infections with CNS involvement treatment with non-arsenic drugs gives rise to an apparent cure since parasites disappear from the circulation but, after a period of weeks, they reestablish themselves in the circulation. The natural immunity of humans to the cattle pathogen T. b. brucei, but not to the morphological indistinguishable human pathogens T. b. rhodesiense and T. b. gambiense, is probably a result of the selective killing of this species by normal human serum containing trypanolytic factors. Unlike in animal trypanosomiasis, the most prominent symptoms of sleeping sickness may result from the marked damage to the CNS in late-stage T. b. gambiense (and T. b. rhodesiense) infections. Melarsoprol and related arsenicals (known for their high systemic toxicity (Trypanocidal Drugs, Man/Drugs Acting on African Trypanosomiasis (Sleeping Sickness) of Humans) are able to cross the blood-brain barrier. A long-term model of African trypanosomiasis in mice producing meningo-encephalitis astrocytosis and neurological disorders can be used to understand the pathogenesis of human African trypanosomiasis from initial infection to advanced stages and to evaluate drug efficacy in the late stage of disease. Trypanocides may also be suitable tools in diagnosis of chronic (subpatent) T. c. congolense infections in cattle. For this purpose drugs are applied intravenously before and in combination with the indirect fluorescent antibody test. Rapid flushing of cryptic trypanosomes from the microcirculation may lead to increase of jugular parasite concentrations within 6–10 min of the administration of diminazene, pentamidine, or homidium chloride. However, diamidines given by the intravenous route are liable to give rise to hypotension and other severe, alarming reactions, some of which are due to histamine release.
Measures currently used to control trypanosomiasis are diagnosis and treatment, chemoprophylaxis, tsetse fly control or eradication of tsetse flies, and the utilization of so-called trypanotolerant breeds. However, this most challenging task in Africa is complicated and hampered by several specific factors. The number of tsetse flies (and thus the occurrence of disease) fluctuates greatly over periods of several years and makes assessment of the actual risk to which livestock are exposed difficult. In addition, control of trypanosomiasis is hindered considerably by the fact that African trypanosomes are able to establish chronic infections in their mammalian hosts because of their highly developed system of antigenic variation. Individual members of the parasite population change the composition of their surface coat so that variations in the composition of these variant surface glycoproteins (VSGs) allow the parasite to escape the host's immune system. Thus fluctuating parasitemias produced by T. brucei are associated not only with the phenomena of variable antigen type (VAT) but certainly also the potential for regulation of trypanosome growth by environmental factors such as epidermal growth factor (EGF), transferrin and low-density lipoprotein. This makes effective immunoprophylaxis unlikely.
In areas with low tsetse fly density the method of choice for controlling African trypanosomiasis seems to be the eradication of the vector. For the time being the spraying of insecticides dominates in tsetse fly eradication. In regions with very low levels of infestation, e.g., by riverine tsetse species (Glossina palpalis group, e.g., G. palpalis, G. fuscipes), trypanosomiasis can be controlled by surveillance and treatment only. Nevertheless, flies of the savanna (and thicket) group (G. morsitans group, e.g., G. morsitans, G. pallidipes, G. austeni) may give rise to severe trypanosomiasis in susceptible stock even if their numbers are low. In these areas commercial cattle ranching may be possible under chemoprophylactic protection. However, tsetse fly density and thus contact between cattle and vector must be reduced by additional spraying of insecticides with residual effects (e.g., synthetic pyrethroids) and by setting up impregnated traps and screens. In areas with medium tsetse fly density the further exploration and logical exploitation of trypanotolerant cattle, including crossbreeding trials with European breeds to increase milk and meat productivity of indigenous trypanotolerant cattle, may offer a realistic alternative to not yet available vaccination. At least in areas with high tsetse fly density even trypanotolerant animals may not survive unless they are treated prophylactically against trypanosomiasis. The control of the disease in fully susceptible stock even under chemoprophylaxis seems to be impossible in regions heavily infected with tsetse.
Tsetse flies can detect odors by means of receptors on their antennae. Experience with insect pheromones was used to identify the chemical components of the ox odor, which might attract tsetse flies and led to the discovery of 1-octen-3-ol. It proved highly attractive to flies of the savanna (G. pallidipes and G. m. morsitans). Thus live bait (e.g. cattle treated with insecticides: spot-on, pour-on), fly traps, and screens impregnated with “essence of ox” and pyrethroid insecticides (e.g., deltamethrin, aplphametrin, or cyfluthrin), and sophisticated ground spraying technology may markedly reduce tsetse infestation in limited areas of riverine woodland or transitional forest-savanna zones. Traps baited with acetone and 1-octen-3-ol have been used in Zimbabwe, Zambia and Malawi to detect the presence and distribution of tsetse flies. It has been shown that isometamidium is capable of eliminating the insect vector form of T. v. vivax. This experimental finding may be of potential significance in the control of trypanosomiasis in the field, particularly in the operation of the sterile insect technique (e.g. in Nigeria).
Effects of infections on vector survival are of interest for the evolution of parasite-vector interactions since trypanosome transmission depends strongly on vector survival and the frequency of genetic factors controlling vector susceptibility depending on the fitness of infected vectors. In several species of tsetse flies, males from natural populations, and from laboratory-bred colonies, are more likely to develop mature trypanosome infections than females.
Today, there is neither a breakthrough in biological control of tsetse flies nor are there promising solutions for a vaccine against African trypanosomes.
The term `trypanotolerance' means reduced susceptibility to trypanosomiasis and denotes an inherited biological property allowing animals to live, breed, grow and survive in a naturally infected environment without exhibiting clinical signs of trypanosomiasis after harboring pathogenic trypanosomes.
In regions where eradication of the vector is not possible with present methods, genetic improvement of trypanotolerant breeds should be attempted. Attention has recently focused on genetic resistance and various selection programs are being discussed to select trypanotolerant animals. Such programs could involve selection of trypanotolerant animals under natural challenge or selection of marker traits (e.g., aspects of the immune response). Selection could also act on polymorphic loci that may affect trypanotolerance, and may be closely linked to genes acting upon tolerance via marker loci. Trypanotolerance is found not only in cattle (all dwarf semiachondroplastic West and Central African types) but also in sheep, goats, and in some rare pony types, such as the Kotokoli of the Ivory Coast. The NŽDama (Hamitic Longhorn of the Bos taurus type as well as those breeds of the West African Shorthorn) is a West African breed (e.g., Gambian cattle) noted for its small size and its trypanotolerance. This humpless breed responds very well to improved management and can attain levels of productivity comparable to that of many African beef breeds of the Bos indicus type, such as the West African Zebu, the Orma Boran, the Ankole, or the Afrikander. In addition the NŽDama can maintain reasonable production levels under conditions of poor management, climate, nutrition and high tsetse fly densities. Trypanotolerant breeds of Zebus, sheep and goats may also exist in East Africa. Field studies on two types of large East African Zebu (Bos indicus) Boran cattle on a beef ranch in Kenyo have demonstrated that a boran type bred by the Orma tribe had a superior response to tsetse fly challenge compared to an improved Boran when introduced to a new locality. Superior resistance to tsetse fly challenge was evident by lower trypanosome infection rate, and when this was untreated, by lower anemia and decreased mortality.
Following the successful feeding of tsetse flies infected with T. c. congolense, cattle develop local reactions of delayed onset (commonly called a chancre) that persist for several days. The proliferation of the parasite in the hosts skin prior to its passage into the bloodstream via draining lymphatics plays an important role in the induction of immunity, as it is only after regression of the chancre that cattle are immune to tsetse-transmitted homologous challenge. Attempts to induce skin reactions by intradermal injection of bloodstream forms of T. c. congolense have failed. Thus, the induction of immunity to trypanosomes may be adversely affected if trypanocidal drugs are given prior to regression of the chancre. In an area of medium tsetse fly challenge it was found that the degree of immunity was greatest in cattle in which infections were established and clinical disease could develop before treatment. Conversely, no immunity developed in cattle treated immediately trypanosomes were seen in the peripheral blood and prior to any evident clinical signs. Induction of immunity to T. c. congolense in rabbits by infection and treatment with homidium chloride may also be adversely affected if animals are infected concurrently with antigenically different stocks of trypanosomes. There was no marked cellular proliferation in the skin at the sites of secondary infection bites following feeding of a single G. morsitans; a chancre failed to develop. Possibly the impaired response was due to drug action preventing trypanosomes from developing extravascularly.
On the other hand, the apparent duration of drug protection has been thought to be influenced by protective immunity which may develop as a result of interactions between insect vector, host, trypanosome population, and drug. These interactive effects may lead to “non-sterile immunity” or “tolerance” in cattle following drug administration and trypanosome challenge. The role of immune responses has been investigated in isometamidium treated Boran cattle under single or repeated challenge with T. c. congolense infected tsetse flies. Six months after treatment two-thirds of the cattle were resistant to challenge, irrespective of whether animals had received single or multiple challenge. The animals had no detectable skin reactions at the site of deposition of metacyclic trypanosomes and produced no trypanosome-specific antibodies, indicating that drug residues effectively inhibited trypanosome multiplication in the skin and thus subsequent parasitemia. It was concluded that immunological priming of the host had not occurred, and that the protection achieved was not related to the development of immune responses by the host enhancing the length and potency of protection afforded by isometamidium. These findings indicate that development of immunity is not necessary for successful maintenance of cattle in tsetse fly areas provided close control of drug regimes is maintained. The results may also indicate that it is essential to allow multiplication of parasites prior to drug treatment to induce immunity in the host.
Induction of non-specific host defense (e.g., macrophage functional activity) by immunomodulators was demonstrated in 1979 by Murray et al. Bacillus Calmette-Guérin (BCG) and Corynebacterium parvum were found to enhance the immune response to T. c. congolense infections in susceptible A/J mice and more resistant C57B1/6J mice, both showing reduced parasitemias and increased survival times. This effect could not be transferred from treated to untreated mice of the identical strain by spleen cells or serum. It is not yet clear by which mechanisms immunomodulators influence the course of infection. The development of effective, immunostimulants may provide attractive, complementary tools for combating trypanosomiasis and should be considered as an additional approach to the complex undertaking of a screening program for new trypanocidal drugs and breeding programs for trypanotolerant livestock.
For animal trypanosomiasis no new drugs of any kind have appeared in the field since the introduction of isometamidium in 1961. Nevertheless, aromatic diamidines continue to provide new compounds of high intrinsic activity. Among these, several compounds are highly active on T. c. congolense and T. v. vivax, while others show a high activity on trypanosomes of the subgenus Trypanozoon. Unfortunately, resistance to one trypanocidal diamidine appears to confer resistance to all diamidines and diminazene-resistant trypanosomes have been shown to be resistant to DAPI (4Ž, 6-diamidino-2-phenyl-indole) and other diamidines synthesized by Dann and his colleagues. Aromatic diamidines (e.g. pentamidine, diminazene) not only inhibit the growth of protozoans but also of bacteria, fungi and tumor cells, generally at concentrations below those found to be active on the host. DAPI forms fluorescent complexes with double-stranded DNA and is now used for the fluorescent staining of prokaryotic and eukaryotic cells. The drug seems to interact with A-T-rich regions of DNA and thereby to suppress the DNA-directed RNA and DNA polymerases. Several trypanocides (quinapyramine, pentamidine, diminazene aceturate, and isometamidium) and a babesiacidal drug (imidocarb) have been investigated in an activated DNA-directed DNA synthesis assay system catalyzed by T. b. brucei DNA polymerases, murine thymus DNA polymerase alpha, and Rauscher murine leukemia virus reverse transcriptase. From the results obtained it was suggested that trypanosomal DNA polymerases are not the selective target of drugs as they showed a similar dose dependent inhibition to other DNA polymerases of eukaryotic cells. Stimulation of reverse transcriptase activity was observed in the presence of quinapyramine and imidocarb but this could be negated by the presence of spermine in the reaction mixture. As part of studies on N-oxidative biotransformation of amidines, potential metabolites of pentamidine have been synthesized. Although several amidoximes of pentamidine and diminazene proved highly active against various African trypanosomes in mice, their potency was inferior to that of the parent compounds. Several compounds of a series of aryl bisbenzimidazoles have shown excellent activity against diminazene-resistant T. c. congolense, T. v. vivax, and T. b. evansi strains. Unfortunately this series caused delayed toxicity in calves, including serious liver and kidney damage.
Several antitumor antibiotics have revealed unsuspected high activity against trypanosomes in vitro, particularly DNA and RNA synthesis inhibitors such as 5-chloro-puromycin. Daunorubicin, an anthracycline antibiotic intercalating with DNA, which is one of the most potent trypanocidal agents in vitro, has proved totally inactive against T. b. rhodesiense in infected mice. Limitations of efficacy and problems with toxicity impose severe limitations on the usefulness of antitumor drugs as potential leads to new trypanocides in humans and animals. The antifungal nucleoside antibiotic sinefungin, which strongly inhibits S-adenosyl-methionine dependent transmethylation reactions, has a marked effect on African trypanosomes in mice when administered intraperitoneally. Goats infected with T. c. congolense and treated with intramuscular doses of 10 or 20 mg/kg b.w. showed relapse of infection; higher doses (up to 50 mg/kg b.w.) were toxic and caused death. Among a series of novel purine derivatives (phosphonylmethoxyalkylpurines and pyrimidines) with antiviral activity against a broad spectrum of DNA viruses some of them showed potential activity in vivo against T. b. brucei at dosages that were below those toxic for mice. Ketoconazole and related azole derivatives with high activity against T. cruzi infections in mice have proved ineffective against T. b. brucei in mice. Among a series of phthalanilides and related compounds, BW 458 C was the most effective in curing short-term and long-term T. b. brucei infections in mice. Cure rates greater than 90% were achieved with the drug at 10 or 25 mg/kg body weight. None of several compounds of a series of suramin analogues was more active than suramin against macrofilariae of Dipetalonema viteae and various Trypanosoma spp. Inhibition of lipid metabolism in the trypanosomes may be central to the therapeutic effects of the garlic extract containing diallyl-disulfide (DAD). DAD is known to have a lipid-regulatory effect and a sulfur-rich compound that readily undergoes ionic interaction with SH being a vital component of coenzyme A. The latter is required in growing cells for the provision of activated acetate molecules, which are then channelled into lipid synthesis and other vital cellular processes.
Salicylhydroxamic acid (SHAM), a substituted aromatic hydroxamic acid, inhibits aerobic energy production (L-glycerol-3-phosphate oxidase system) in trypomastigote stages; it can clear temporarily bloodstream infections of T. b. brucei in rats if administered concomitantly with glycerol. In practice only one far from ideal drug, melarsoprol (Mel B) is available to treat late-stage sleeping sickness. Calcium (Ca) has a synergistic effect on this trypanocide and has been shown to be more critical in its action than SHAM +glycerol. These data may be important in the clinical management of sleeping sickness. If total Ca is reduced in a patient it is possible that supportive therapy to restore Ca concentrations could improve the therapy, especially in late-stage Gambian infections.
The reason that potent chelators are trypanocidal but not toxic to mice may relate to acute competition for Fe between the host's Fe-binding proteins like transferrins and ferritin and the parasite's Fe requirement. Several chelators such as caffeic acid, cuproine, and other commercially available chelators, which had shown heme sparing or inhibition of growth of Crithidia fasciculata in vitro were active against T. b. rhodesiense in mice after high doses only. Divalent cation chelators such as ethylenediamine tetraacetate (EDTA) or the calcium-specific chelator ethyleneglycol tetraacetate (EGTA) can abolish the synergistic action of heparinized rat blood with SHAM +glycerol. Transferrin may also function as a drug carrier in African trypanosomes in such a manner that complexes of transferrin with isometamidium (Samorin) are targeted directly with high specificity into the lysosome system of T. c. congolense.
DL-α- difluoromethylornithine (DMFO = eflornithine) is a selective and irreversible inhibitor of ornithine decarboxylase and a key enzyme in polyamine biosynthesis in T. b. brucei. The substituted amino acid was shown to have activity against CNS T. b. brucei infections in rodents and is the only `new' drug to be developed for the treatment of sleeping sickness in humans. It has proved to be an effective treatment for late stage infections of T. b. gambiense in humans (Trypanocidal Drugs, Man/Drugs Acting on African Trypanosomiasis (Sleeping Sickness) of Humans).
There are various areas considered as leads in research relevant to the development of potential new agents for African trypanosomiasis and targets for chemotherapeutic attacks such as, glycolytic enzymes (non-oxidative branch of pentose phosphate pathway = PPP), antigenic variation, and trypanothione metabolism in trypanosomes. Oxidative branch of PPP might be an alternative lead for new drugs. It maintains a pool of NADPH (reduced form of nicotinamide adenine dinucleotide phosphate required for synthesis of fatty acids via phosphogluconate pathway) that serves to protect against oxidant stress and which generates carbohydrate intermediates used in nucleotide and other biosynthetic pathways. Thus 6-phosphogluconate dehydrogenase (6PGDH) in T. b. brucei may be a potential target for chemotherapy because in other eukaryotic organisms the deletion of the gene encoding 6PGDH is lethal. The gene encoding T. b. brucei 6PGDH has been cloned, and the enzyme purified and crystallized. Suramin inhibits 6PGDH, and trivalent aromatic arsenoxides inactivate the enzyme with marked potency. Considerable attention has been devoted also to topoisomerases of kinetoplastid organisms. This group of enzymes could be another valuable drug target for new trypanocides. Topoisomerases, which mediate topological changes in DNA, are essential for nucleic acid biosynthesis and for cell survival. Topoisomerase II activity has been purified from Leishmania donovani, T. cruzi (Trypanocidal Drugs, Man) and T. equiperdum, and topoisomerase II genes have been cloned also from T. b. brucei and T. cruzi. Studies with purified topoisomerases indicate that the enzymes from kinetoplastids generally exhibit the expected inhibitor sensitivities. Thus activity is reduced by intercalators acting by deforming the DNA substrate, minor groove binders (compounds that bind in the minor groove of the DNA helix) and compounds that compete for binding at the enzyme's ATP site (e.g., novobiocin, coumermycin). Agents that specifically inhibit type II enzymes by trapping the enzyme on its DNA substrate, forming a `cleavable complex', are the fluoroquinolines and etoposide. Thus, antibacterial fluoroquinolines were shown to exhibit marked activity in vivo against Leishmania donovani. Some classical trypanocides such as DNA-binding agents (diminazene and pentamidine: minor groove binders) and intercalators (e.g., ethidium bromide) are well known for their ability to generate dyskinoplastic trypanosomes, which retain mitochondrial membranes but lack detectable kDNA. Selective inhibition of mitochondrial topoisomerase II may be an explanation for the propensity of these drugs to induce dyskinoplastic cells. Because kDNA is not essential for the survival of bloodstream form of African trypanosomes, nuclear rather than mitochondrial topoisomerases should be the preferred target for drug search. Differences in parasite and mammalian topoisomerases may provide the basis for selective toxicity of new trypanocidal compounds. On the other hand, kinetoplasts can be an obligatory target for antitrypanosomal drug action if these organelles are important for successful subsequent cycling into the insect vectors. There must be some mechanism to assure that an organism does not replicate the nucleus and divide until or unless it has replicated its kinetoplast. Drug targeting of kinetoplasts, then, could interfere with cell replication by preempting this regulatory mechanism. Thus identification of regulatory mechanism generating dyskinetoplastic resistance in trypanosomes could possibly provide a basis for new therapeutic approaches. Also molecular biological investigations, as well as inducible gene expression systems (e.g., the tetracycline-responsive repressor of Escherichia coli, TetR) in trypanosomes, could suggest potential targets for chemotherapy and pathogenicity.
For cattle treatment only a few drugs have been developed, and these are involved in resistance problems today. Under such conditions (such as in cancer chemotherapy) exploration of combinations might be an alternative strategy. Therefore, the possibilities of trypanocidal synergic effects of known drugs have been extensively examined in vitro and in vivo using monomorphic laboratory strains of T. b. rhodesiense. Only suramin +tryparsamide, suramin +puromycin, suramin +diminazene, and 9-deazainosine +DL-α-difluoromethylornithine (cf. also Search for New Drugs) have been shown statistically significant synergy. Another example of a successful combination therapy is the suppression of chronic T. b. brucei infections in mice (CNS involvement) by diminazene diaceturate or suramin, each combined with a substituted 5-nitroimidazole (e.g., fexinidazole or MK 436). None of these drugs administered singly caused 100% permanent cure. Only fexinidazole (Hoe 239) was able to cure a high percentage of the mice when given repeatedly at relatively high dose levels of 250 mg/kg. In several experimental studies fexinidazole has also been found to exhibit a strong effect against T. cruzi, T. vaginalis and E. histolytica in vivo.
Although there are different ways of combating cattle trypanosomiasis, each of the control methods in use at present has serious limitations. Because of the major economic importance of trypanosomiasis in cattle the great majority of control measures have been aimed primarily at the protection of these animals by the use of suitable trypanocidal drugs. In the absence of a suitable vaccine, chemotherapy and chemoprophylaxis are the most important tactics, which are available as part of any strategy of trypanosomiasis control. They are still considered to be the most effective measures for trypanosomiasis control.
Drugs used for the treatment and prophylaxis of animal trypanosomiasis center on a small number (Table 1). They can be characterized on the basis of their ionizaton at blood pH as cationic or anionic drugs. Cationic drugs are quaternary ammonium trypanocides (quinapyramine, homidium, pyrithidium, isometamidium), and aromatic diamidines (diminazene and pentamidine). The only anionic drug currently in use is presented by suramin. It is a sulfated naphthylamine derivative that readily binds to plasma proteins; it is still widely used in the treatment of equine trypanosomiasis.
The risk of infection to which cattle are exposed is closely related to the density and the species of tsetse fly present. The incidence of tsetse flies thus chiefly determines the frequency of treatment, which is in most regions regulated by the government. The nomadic habits of the major cattle-owning peoples have given rise to the widespread use of trypanocidal drugs and, in general, treatment of individual animals is not practiced. Ormerod (1979) pointed out that rationale for treating cattle trypanosomiasis is entirely different from that for treating sleeping sickness, for the following reasons. (1) Trypanosomes are very much more common, so that any animal, which becomes infected by tsetse flies, is liable to be infected. Therefore treatment of individuals (or even herds) has no general sanitary significance. (2) Drugs are often given prophylactically to cattle on their way to slaughter in Africa. Cattle are usually moved over long distances to provide meat in urban areas and prophylactic drugs are administered so that animals can pass through the “fly belt”. Prophylactic treatment is particularly afflicted with problems concerning variations in the length of protection resulting from varying field situations and the rate of drug elimination from the body (preslaughter withdrawal time). The duration of chemoprophylaxis thus not only depends on the degree of tsetse fly challenge but also on the timing of treatment in relation to occurrence of infection. Insufficient drug protection may result if infection by tsetse flies occurs too early in the trek, and cattle may then succumb to infection before reaching their destination.
The period of effectiveness of prophylactic drugs thus varies with environmental conditions, the tsetse fly challenge and activity of the treated animal as well as actual concentration of the active drug (there is the risk of producing a too long-lasting subcurative concentration in blood and tissues). Thus, the period of protection may be considerably reduced particularly during strenuous activity, especially when trade cattle pass through a fly belt in the course of their journey.
The prophylactic action of drugs has been prolonged by preparing complexes of suramin (anionic drug) with cationic/basic drugs, thereby reducing systemic toxicity of the drugs. Although a homidium-suramin complex gave extended protection (6–12 months), it caused unacceptably severe reactions at injection sites. A quinapyramin-suramin complex proved active at a single dose of 50 mg/kg body weight in protecting adult pigs and piglets for at least 6 and 3 months, respectively. However such long acting drugs often cause rapid development of drug resistant trypanosomes. Encapsulation of drugs, either in polymers or in artificial phospholipid membranes (liposomes) has been known for a long time. Preparing a complex of isometamidium with the well-defined polyanion dextran, thereby reducing its toxicity, could enhance the duration of protection produced by the drug. Entrapping homidium bromide in bovine carrier erythrocytes has caused slow release of the drug. However, the various short comings of such preparations, like drug quality problems (standardization), marked drug residues (possibly posing a human health hazard) and severe reactions at sites of injection, have hindered the further preclinical development of such preparations. Recently, more promising results were obtained using different types of subcutaneously implanted slow release devices (SRD) containing polycaprolactone/homidium bromide SRD or more readily biodegradable poly (D, L-lactide) or poly (D, L-lactide-co-glycolide) SRD containing either isometamidium chloride or homidium bromide for intramuscular administration. As a result local toxicity was minimized and prophylactic effects in comparison with the parent compounds were markedly prolonged. When breakthrough isolates derived from SRD-treated animals (rabbits) were compared with the original T. congolense strain, such isolates showed some loss of sensitivity to homidium only.
The conditions under which `man made drug tolerance' develops in the field are derived basically from under-dosing due to incorrect estimation of body weight; this is difficult to avoid when mass treatment is involved. A high incidence of trypanosomiasis in conjunction with the irregular use of prophylactic and therapeutic drugs also favors the emergence of drug-resistant trypanosomes. Thus, drug-resistant parasites may emerge in any situation where prophylaxis and therapy are inadequate for the degree of tsetse fly challenge. This may be the case particularly in regions of high tsetse fly challenge. The misuse of drugs leads consequently first to “individual” resistance and then to “area” resistance. Generally, prophylactic drugs induce resistance more rapidly in trypanosomes than do `therapeutic' drugs. The latter drugs reach trypanocidal plasma levels relatively quickly and may be more rapidly metabolized and excreted from organisms than `prophylactic' drugs. In areas with a high incidence of tsetse flies, subcurative drug levels may already exist towards the end of the protection period, and this is the case particularly after using drugs for prophylaxis. Treatment must therefore be repeated to restore trypanocidal plasma concentrations.
The phenomenon of `natural (intrinsic) drug tolerance', i.e., variation in drug sensitivity that is not dependent on previous exposure to the drug concerned, has been demonstrated in T. v. vivax and T. c. congolense. Thus, West African T. v. vivax strains seem to be more susceptible to homidium than are East African T. c. congolense strains. By contrast, T. c. congolense strains appear to be more susceptible to diminazene than are T. v. vivax strains. It is likely that the initial appearance of homidium-resistant T. c. congolense strains and diminazene-resistant T. v. vivax strains can be connected directly with the varying intrinsic sensitivity of these species of a given drug. Some of this variation in drug sensitivity may also be the result of persistent cross-resistance induced by quinapyramine, which was extensively used for therapeutic and chemoprophylactic treatment before homidium became the drug of choice. Differences in drug sensitivity of stocks of the subgenus Trypanozoon have also been reported. In an in vivo assay designed to minimize the influence of host-parasite interactions using X-irradiated trypanosomes, it was demonstrated that isoenzymically defined West African T. b. brucei stocks were not as sensitive to pentamidine and diminazene as typical East African stocks. This test sought to measure the intrinsic sensitivity of a trypanosome population by reducing the influence of extrinsic determinants of drug sensitivity, in particular trypanosome “penetration” of tissues inaccessible to drugs and host antibody-mediated relapses of parasitemia.
Problems involved in using inappropriate in vivo models for testing drug sensitivity may be overcome by culturing trypanosomes in vitro, allowing precise detection of intrinsic sensitivity of all stages in the life cycle of trypanosomes. The use of simple in vitro assays using feeder layer-free in vitro systems may help to obtain rapid information on the susceptibility of isolated trypanosome strains to the drug concerned. However, based upon the ability of these assays to predict potential drug efficacy in vivo, not all fresh isolates or clones of trypanosomes can be grown in feeder layer free systems. Thus, a combined mammalian feeder layer-trypanosome culture system may make it possible to determine different effects of a compound on host cells (general toxicity) versus parasites (selective toxicity). Calcium antagonists of several chemical classes including verapamil, cyproheptidine, desipramine and chlorpromazine, alone and in combination with various trypanocidal drugs (suramin, diminazene and others), were unable to reverse resistance in T. evansi to any of the trypanocides tested in vitro. These results are in contrast with those occurring in T. cruzi, Plasmodium, Leishmania and cancer cells, in which calcium antagonists have successfully reversed resistance.
Current limitations on drug efficacy are due to the occurrence of trypanosomes showing multiple drug tolerance to several drugs with close chemical relationship. This has been true also for a T. v. vivax strains first reported from Kenya in 1985. Thus a `cocktail' of 11 T. v. vivax isolates has proved resistant to all drugs on the market, e.g., to isometamidium chloride (2 mg/kg b.w.), diminazene aceturate (3.5 mg/kg b.w), homidium chloride (2 mg/kg b.w), and quinapyramine sulfate (5 mg/kg b.w.). This finding appears to have implications of considerable importance to East African cattle producers. The ability of T. v. vivax to cause a hemorrhagic syndrome has also been discussed. Hemorrhages apparently do not occur in all cases and not in all stages of the disease. This form of trypanosomiasis can be acute or peracute and is responsible for severe losses in unprotected stock (for more information on hemorrhagic T. v. vivax infections cf. Use of Drugs in the Field to Control Cattle Trypanosomiasis).
The main problem in chemotherapy and chemoprophylaxis is to control the widespread cross-resistance in trypanosomes to the few drugs on the market (Table 1). Resistance to a drug, which has developed as a result of previous exposure of trypanosomes to a different drug of the same series or to a drug of an unrelated series can only be effectively controlled by using drugs that do not induce resistance to each other. If this is the case, then they can be used alternately when resistance to either drug appears in the field. Already in the early 60s significant knowledge of cross-resistance patterns was obtained from studies of large numbers of cattle maintained under controlled field condition in East Africa. Insufficient response of trypanosomes to certain prophylactic and curative drugs at recommended doses led to the strategic use of `sanative' pairs of drugs in the field. Such drug pairs include homidium/diminazene and isometamidium/diminazene, which show no cross-resistance although quinapyramine-resistant trypanosomes confer resistance to each of these drugs. Moderate side-resistance may also be present between homidium, pyrithidium and isometamidium, which belong to the same chemical class of phenanthridines. Increased doses of isometamidium (1–2 mg/kg b.w.) may, however, control resistance to homidium and pyrithidium.
In curative field programs homidium may be used until evidence of resistance appears. It should then be replaced by diminazene, which generally controls infections in cattle reinfected with homidium-resistant parasites. Homidium may be used again after a year or so. Isometamidium and diminazene may be used alternately in prophylactic field programs. However, the appearance of drug-tolerant strains is believed to be inevitable in these programs, particularly in high-risk areas where isometamidium chloride is used at the standard dose of 1 mg/kg b.w. every 3 months. This dose may protect cattle against trypanosomiasis for 6–12 weeks if tsetse fly challenge is not too high. Higher dose levels can cause local reactions, a problem, which is common to all prophylactic drugs currently used (for comments see Table 1).
Control of the disease has been maintained when quarterly prophylactic injections with isometamidium were supplemented by block treatment with diminazene at regular intervals, i.e., every 6 months, 1 month prior to routine treatment with isometamidium. `Sanative' diminazene will not control the situation if the challenge becomes too high as a result of increasing rates of reinfection with resistant trypanosomes. T. v. vivax and T. c. congolense strains, which survive isometamidium doses of 1 mg/kg b.w. and are cross-resistant to homidium can be controlled, however, by repeated administration of diminazene aceturate at a dose of 7 mg/kg b.w.
Field observations on the stability of drug-resistance in trypanosomes undergoing cyclical transmission are contradictory. Some observations suggest that drug resistance is stable and transmissible, while other investigators have assumed that drug-resistance in a trypanosome population is transient in the absence of drug pressure and infected cattle. In a series of experiments drug tolerance to curative doses of trypanocides was shown to be of stable nature, while T. v. vivax and T. c. congolense were transmitted through tsetse and cattle. However, it was assumed, that in the field competition between resistant and sensitive parasites in the trypanosome population might lead to an advantage for sensitive forms resulting in a gradual disappearance of drug-resistant parasites.
There has been increasing public health concern about the consumption of trypanocidal drug residues in foods. A survey conducted recently in central Kenya has shown significant quantities of trypanocides in cattle meat from various slaughterhouses. Previously, the phenantridines (Table 1) isometamidium (for MRLs see Table 1) and quinapyramine have been believed generally to maintain trypanocidal blood concentrations for longer periods than diminazene. This led to the assumption that storage in and release from deep compartment are due to a process different from that occurring with diminazene. Thus, diminazene has been found to have only a limited prophylactic effect, and patent parasitemia has often been detected 2 weeks after treatment. This indicates that diminazene may be rapidly removed from bloodstream if given as the readily water-soluble aceturate. In contrast, the virtually water-insoluble diminazene dihydrochloride (or embonate) yielded in rats a fairly long protection period of 56–70 days at subcutaneous doses of 1x 16.5 and 1x 33 mg/kg b.w. against a high challenge of T. b. rhodesiense and T. b. gambiense. It was also demonstrated that diminazene diaceturate was “rapidly” removed from the plasma in mice whereas its tissue concentration remained relatively high for several weeks. In rhesus monkey (Macaca mulatta) the elimination of diminazene aceturate (single intramuscular dose of 20 mg/kg b.w.) occurred in two phases with half-lives of 2.1–2.7 h and 15.5–23.3 h; the protection period against a high challenge of T. b. rhodesiense was 21 days. Similar biphasic elimination of the drug was observed in rabbits after intramuscular injection of 3.5 mg/kg b.w. Seven days after treatment 40%–50% of the dose had been excreted in the urine and 8%–20% in the feces; the highest diminazene residues were found in the liver and corresponded to 35%–50% of the dose given.
Pharmacokinetic studies in cattle provided further evidence for the validity of a two-compartment model in the case of diminazene; there were a biphasic profile and two phases of distribution. Pharmacokinetic properties of diminazene diaceturate [bisphenyl-U14C] (3.5 mg/kg b.w. i.m.) were investigated in healthy calves. Levels of radioactivity were determined in the blood, plasma, urine, feces, and edible tissues. There was a rapid onset of absorption, which led to high blood, and plasma levels (4.6 nEq/ml). The decrease in concentration followed a biphasic process with half-lives of 2 and 188 h; 20 days after administration 72.2% and 10.3% of the dose had been excreted in the urine and feces, respectively. The main product in urine was unchanged diminazene. Radioactivity could be detected in blood and plasma for up to 20 days after administration. Distribution studies revealed low concentrations in edible tissues, particularly in skeletal muscle and fat. From these results it was concluded that diminazene is not as rapidly and entirely metabolized (or biotransformed) in the body as suggested previously. Following the results a preslaughter withdrawal time of 21 days for all edible tissues (also liver) was recommended for cattle. A similar long preslaughter withdrawal period (14–20 day) was estimated for sheep after a single intramuscular dose of diminazene 3.5 mg/kg. Drug concentrations were determined in plasma and equilibrium dialysis and high-performance liquid chromatography. As expected, dairy goats that had received two successive intramuscular doses of diminazene aceturate 2 and 3.5 mg/kg b.w. showed somewhat different pharmacokinetics from those that received a single injection. The estimated preslaughter withdrawal period was between 28 and 35 days. Dairy cows repeatedly infected with different strains of T. congolense and treated with different dose of radiolabelled diminazene aceturate have been investigated for dependence of drug residue levels in milk. Results of this study indicate that the degree of parasitemia (anemia) affects the distribution, disposition, and elimination of diminazene. At 3.5 mg/kg b.w. 0.4% of the dose was excreted in milk after 21 days, while 0.54% of the 7 mg/kg b.w. dose was excreted during the same time. On the basis of data of half-lives for the second phase (elimination phase) milk from treated animals should not be consumed for at least 3weeks posttreatment. In rabbits treated with a single intramuscular dose of [14C] homidium bromide 1 or 10 mg/kg b.w. blood and tissue levels reached a maximum within 1 h then fell rapidly. After 4 days 80%–90% of the radioactivity injected had been excreted, 33% in the urine and 66% in the feces. In view of the rapid rate of drug excretion it was assumed that the time and level of infection relative to the time of drug administration might markedly affect the protective action of ethidium. Some doubt must therefore remain about the value of this drug for the prophylactic treatment of slaughter cattle as recommended previously.
Based on a review of the literature with special reference to control of animal trypanosomiasis in Africa, the conclusion was that each of the control methods in use has serious limitations. This appears to be true also for the present situation. Today and in the past, the effectiveness of chemoprophylaxis and chemotherapy has been reduced markedly by the widespread development of drug resistance. The enormous cost involved in research on and development of new drugs, which industry has to consider, have meant that very little research on potential trypanocides has been done. Thus, 20 years ago some pharmaceutical companies were still involved in research on the chemotherapy of trypanosomiasis, despite the financial considerations of the relatively small market and uncertain financial returns. Economic and ecological constraints on trypanosomiasis control are still evident, and the high cost involved in a continuing program of eradication of tsetse flies, or even of isolated tsetse-belts, is often beyond the reach of individual countries. Furthermore, tsetse fly clearance remains an unreliable control measure when continued surveillance is not guaranteed, and tsetse fly eradication will not necessarily result in the eradication of trypanosomiasis since T. v. vivax and T. b. evansi can cause infections without cyclical transmission and can be spread mechanically by biting dipterans. On the other hand, attempts to eradicate tsetse flies by chemical control, e.g., by massive aerial insecticide spraying, are always associated with considerable adverse effects on environment.
Biological and genetic control methods are still at an early stage of development (as 30 years ago) and control of trypanosomiasis by immunological tools will only be achievable on a long-term basis. It seems likely that research into the response of trypanotolerant cattle will be of special value although host-parasite relationships and thus, trypanotolerance are still poorly understood. All these limitations would be less important if the present control measures were used in an integrated control program; the importance of international cooperation in combating trypanosomiasis should be stressed. However, the problems encountered in the organization of control programs differ greatly according to whether the method of control is directed against the parasite or the vector tsetse fly. Campaigns directed against the vector are much more a matter of straightforward organization, logistics, and cost (e.g., considerations of the economic return from development after tsetse fly eradication) than are those which involve the attack of infections of the vertebrate host using curative or prophylactic drugs. Perhaps the most valuable use of trypanocidal drugs is in the development of cattle rearing and production in areas where tsetse fly eradication cannot be achieved in the near future. In such areas, conditions can gradually be created under which operations against tsetse flies may be undertaken. Thus, attention must be drawn to one of the chief remaining constraints on the improvement and multiplication of trypanotolerant livestock, i.e., the relatively low reproductive performances of certain cattle breeds under traditional management systems.
