Induction of human serum-sensitive Trypanosoma brucei stabilates into human serum-resistant "T. rhodesiense".

When chronic Trypanosoma brucei infections of mice are treated with 20 mg/kg suramin, those trypanosomes which have escaped chemotherapy because they are residing in the brain, exhibit a higher degree of human serum resistance than the original infection. This resistance increases if the chronic infection is retreated for a second time, before the trypanosomes in the brain are tested by the blood incubation infectivity test. The transformation is not due to a selection of T. rhodesiense from a T. brucei/T. rhodesiense mixture in the original stabilates as cloned derivatives also exhibit these same characteristics. The implications of this finding are discussed.

The use of Erlangen diamidine 98/202 in relapsing Trypanosoma brucei infections in mice.

Mice infected with T. brucei GVR 23/1 or T. brucei GVR 35/1 for 21 days were treated with the Erlangen diamidine 98/202 (6-amidino-2-[4' amidino-phenyl] thionaphthene-dilacate) at either 5 mg/kg or 35 mg/kg. At both these dose levels the parasites were initially cleared from the circulation although the infections eventually relapsed due to reinvasion from the central nervous system. If the 98/202 therapy is followed by treatment with 5-nitroimidazole only a small number of mice are permanently cured. Adverse reactions, especially at the 35 mg/kg dose level, were noted in one experiment. The Erlangen diamidine given to infected mice three days after infection was able to permanently cure both Trypanosoma brucei stabilates.

Treatment with suramin and 2-substituted 5-nitroimidazoles of chronic murine Trypanosoma brucei infections with central nervous system involvement.

Mice infected with either of two isolates of Trypanosoma brucei, GVR 23/1 or GVR 35/1, develop a chronic infection in which trypanosomes are localized in the central nervous system. These infected mice were used to evaluate the efficacy of a combination drug treatment comprising suramin and one of three 2-substituted 5-nitroimidazoles. None of the three 5-nitroimidazoles tested alone, cured mice when administered 21 days after infection. However, it was found that T. brucei GVR 23/1 infections could be cured by a single dose of 20 mg/kg suramin followed by a single dose of 80 mg/kg L611,744 [3a,4,5,6,7,8,9,9a-octahydro-3-(1-methyl-5-nitroimidazol-2yl)cycloocta(D) isoxazole]. The single dose of 20 mg/kg suramin had to be followed by four doses of 80 mg/kg L611,744 to cure mice infected with another stabilate, T. brucei GVR 35/1. A single dose of 20 mg/kg suramin followed either by four doses of 250 mg/kg MK 436 [3a,4,5,6,7,7a-hexahydro-3-(1-methyl-5nitro-1H-imidazol-2-yl)-1, 2-benzisoxazole] or four doses of 70 mg/kg of a dihydroxy analogue of MK 436 [cis-3a,4,5,6,7,7a-hexahydro-3-(1-methyl-5-nitro-1H-imidazol-2-yl)-1, 2-benzisoxazole-6,7-diol] also permanently cured all T. brucei GVR 35/1.

The antibody repertoire in infection and vaccination with Dictyocaulus viviparus: heterogeneity in infected cattle and genetic control in guinea pigs.

The antigen recognition profiles of serum antibody from calves infected or vaccinated with irradiated Dictyocaulus viviparus larvae were analysed by immunoprecipitation of radio-iodinated in vitro-released excretory-secretory materials from live adult parasites. Immunoprecipitates were analysed by SDS-PAGE and considerable heterogeneity in antigen recognition between individual animals was observed, regardless of infection regimen. This heterogeneity was also found to occur amongst outbred guinea pigs infected with the parasite and permitted an examination of the genetics of the effect using inbred guinea pigs (Strains 2 and 13). The antibody repertoires of the two strains were distinct, with only slight variation occurring between individuals within a strain. Previous work on nematode infections in rodents has demonstrated a role for the major histocompatibility complex (MHC) in the control of the immune repertoire. If this, as is probable, holds for the guinea pig, then it can be ascribed to the MHC Class II region because Strain 2 and Strain 13 bear identical Class I alleles but disparate Class II alleles. Whilst there is no evidence to date that the efficiency of vaccination of cattle is influenced by genetic factors, the operation of vaccines based on a single or a few molecularly cloned parasite antigens might be seriously compromised by the kind of genetic restriction to the immune repertoire described here.

Epidemiological aspects of lungworm infections of goats in Morocco.

The results of a survey of lungworm infections in goats in the Middle Atlas and Rabat areas in Morocco during 1990-1992 are reported. Five species were recorded: Dictyocaulus filaria, Protostrongylus rufescens, Cystocaulus ocreatus, Muellerius capillaris and Neostrongylus linearis. The parasitological profile of protostrongylid species was represented by Muellerius (69-78%), Protostrongylus (16-25%) and Cystocaulus (5-6%) in the Rabat and Middle Atlas areas. Neostrongylus was virtually non-existent (under 1%) in both regions. Multigeneric infection involving several species of lungworms reached 54% in Rabat and 88% in Middle Atlas. Dictyocaulus infection of goats does not appear to be a serious problem. Infection rates of 40% and 50%, and average worm burdens of three and five worms per kid and adult goat were recorded in autumn in the Rabat area. A similar pattern was noted in Middle Atlas. In contrast, the incidence of small lungworm infections in goats is widespread at levels likely to be of economic significance. The level of infection was considerably higher than the Dictyocaulus infection and the infection rate was virtually 100% in both age groups in the two areas. The overall worm burdens averaged 77.03 +/- 22.6 parasites per adult goat and 44.16 +/- 16.3 per kid in the Rabat area, whereas the corresponding figures in Middle Atlas were 51.48 +/- 16.65 and 34.06 +/- 2.69 worms. The periods of high risk of infection by small lungworms were autumn, early winter and late spring-early summer. However, the heaviest infection by adult worms and the highest larval excretion were observed in late autumn and winter when molluscs were heavily infected. The periparturient period seemed to exert a positive influence on protostrongylid larval production. The output of first stage larvae (L1) of lungworms was significantly higher in goats than in sheep. Thus, goats may play a greater role in pasture contamination. The epidemiological factors influencing the seasonal fluctuations of lungworms are discussed and a timetable of recommended treatments is suggested.

Some factors influencing the immunisation of sheep with irradiated Haemonchus contortus larvae.

Over the last 6 years a number of experiments in which sheep were successfully immunised with 2 doses of 10 000 Haemonchus contortus larvae, irradiated at 60 krad in a gamma-source, have been reported by the authors. In this paper, the failure of such a regimen of immunisation, using the same strain of larvae, is reported together with an investigation of the possible causes. Technical errors were eliminated as a cause, on the grounds that the failure occurred in 2 separate laboratories using different 60Co sources, as was increased radiosensitivity of the continuously passaged strain of H. contortus as measured by the percentage development of irradiated larvae in 6 naive lambs. From experiments utilising 18 lambs and using both irradiated and normal larvae, it was postulated that the strain had lost its original degree of immunogenicity. Subsequently, in 2 experiments involving 27 lambs, it was shown that irradiation at 40 krad was completely successful in restoring immunogenicity and producing a degree of protection against challenge similar to that previously reported with 60 krad. It was concluded that, with the current strain of H. contortus, 60 krad is now too high a level of irradiation of larvae to produce a consistently high degree of protection against challenge.

Isotype-specific antibody responses to the surface-exposed antigens of adult and larval stages of Dictyocaulus viviparus in infected and vaccinated calves.

The antibody responses to the surface-exposed antigens of living larval and adult Dictyocaulus viviparus were measured by quantitative immunofluorescence using sera from calves infected with, or vaccinated against, the parasite. In infected animals, the surface of the sheath of the third-stage larvae (L3) (retained cuticle of second-stage larvae (L2)) proved highly immunogenic despite the fact that it is thought to be shed prior to parasite penetration of the host intestine. When responses to the surface of exsheathed larvae (L3 cuticle) were measured, a high level of heterophile IgM antibody was detected in the serum of animals that had not been previously exposed to the parasite and, following infection, a specific IgG response was detected against the exsheathed L3 surface. The antibody response, however, was less marked than that observed against the intact L3 sheath. Responses of patently infected animals to the adult surface showed an initial IgM response that was superseded with time by IgG1 and IgG2 responses. Vaccinated animals showed only low level responses to the surfaces of the L3 sheath, L3 cuticle and adult stages following immunisation with two doses of irradiated larvae. The immunised animals produced a strong antibody response to the larval surface antigens following challenge with infective larvae but they failed to produce antibody to the surface of adult parasites. These results show that the surfaces of all the stages of D. viviparus examined are immunogenic in infected calves and, depending on the developmental stage, infection regime, or time of infection, high levels of parasite-specific IgG1 or IgM are stimulated. It has previously been shown that significant levels of protective immunity can be obtained in naive animals following passive transfer of serum from infected calves. Thus, the antibody responses detected in the work reported here may be of relevance in protective immunity against dictyocaulosis.

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.

Parenteral vaccination of calves against the cattle lungworm Dictyocaulus viviparus.

Protection against challenge with Dictyocaulus viviparus larvae was studied in two groups of calves. The first group was vaccinated orally with an irradiation-attenuated larval vaccine on two occasions, 28 days apart, as recommended by the manufacturer. Each dose contained 1000 larvae. The second group was vaccinated by two subcutaneous injections of vaccine, also 28 days apart. Compared with unvaccinated calves the protection in both groups of vaccinated calves was similar, ie, over 95 per cent reduction in adult worm burdens after an oral challenge of 3000 to 4000 larvae. These results indicate that the passage of irradiated larvae through the intestine and mesenteric lymph nodes is not necessary for the stimulation of a high degree of immunity and opens up the possibility of parenteral vaccination against this and related diseases.

Interference between drug-resistant and drug-sensitive stocks of Trypanosoma congolense in goats.

A study was undertaken in goats to investigate the ability of two unrelated stocks of Trypanosoma congolense, one of which is highly sensitive to isometamidium chloride and one which is drug-resistant, to become established in the presence of an existing infection with the other stock. The goats, which were initially infected with the sensitive strain and were then challenged with the resistant strain, were cured by treatment at 0.1 mg kg-1 isometamidium, indicating that the resistant stock did not establish an infection. Goats initially infected with the resistant stock, which were then challenged with the sensitive stock, experienced temporary remission of infection followed by relapse after treatment at 0.1 mg kg-1 isometamidium. In contrast, the goat infected only with the resistant stock remained parasitaemic following treatment at 0.1 mg kg-1. This suggests that superinfection with the sensitive stock resulted in the establishment of infection, which suppressed the resistant stock to below the limit of detection of the method used. These observations suggest that isometamidium-resistant stocks may be less viable than sensitive strains, and could explain the relative scarcity of isometamidium resistant in the field.

Theileria parva: the immune status of calves born of dams immunised against East Coast fever.

Eight Friesian cross cows three months pregnant to a single Friesian bull were immunised against East Coast fever by infection with Theileria parva (Muguga) sporozoite stabilate and treatment with pyrrolidino-methyl tetracycline. They were challenged with the homologous stock four times before calving and a fifth time after calving, and resisted all five challenges which killed all of the five groups of five susceptible controls. Calves born to these hyperimmunised dams were fully susceptible on challenge with the same stabilate, as were susceptible cows from the same farm and their calves. In both instances the calves died three to seven days earlier than the cows which were approximately 10 times heavier. These results show that one- to two-month-old taurine calves from artificially immunised dams are not protected from experimental T parva sporozoite challenge and that there is no inherent calfhood resistance to East Coast fever.

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.

Attempted protection of zebu cattle against trypanosomiasis using a multi-stabilate vaccine.

The protective value of double vaccination with 11 recently isolated stabilates of Trypanosoma congolense given either as live or dead organisms, followed by trypanocidal therapy, was assessed in zebu cattle subsequently challenged by nine of the original stabilates. Both vaccination regimens failed to prevent the animals becoming parasitaemic and eventually succumbing to the infection, although the mean survival times and prepatent periods of the calves which received the live vaccine were longer than those of both the groups which had received the dead vaccine and the challenge controls.

Response of sheep to experimental concurrent infection with tick-borne fever (Cytoecetes phagocytophila) and louping-ill virus.

The pathogenesis of concurrent Cytoecetes phagocytophila and louping-ill virus infection was studied in two experiments. In the first experiment 18 four- to seven-year-old rams were used. Ten were infected with C phagocytophila and five days later eight of these animals and the remaining eight sheep were infected with louping-ill virus. The two rams infected with C phagocytophila alone developed no clinical signs apart from a transient pyrexia, while only three of the eight rams infected with louping-ill virus alone showed mild clinical signs. In marked contrast, all eight dually infected sheep developed severe clinical signs with pronounced depression and dysentery and three died and five were killed in extremis. They developed higher titres of viraemia and the antibody response was depressed while necrotising lesions affecting a variety of organs were detected at post mortem examination. Rhizomucor pucillus was recovered from these lesions in seven of the eight sheep. A second experiment using 10 sheep, five aged seven months and five aged two to three years, confirmed the findings of the first experiment indicating that the age of the animal had not significantly influenced the initial result. It was concluded that C phagocytophila infection could enhance the pathogenicity of louping-ill virus and that, operating together, the two pathogens facilitated fungal invasion. It is postulated that sudden deaths in sheep recently transferred to tick-infested pastures may be due to this newly described syndrome.

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.

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.

Control of parasitic bronchitis in calves: vaccination or treatment?

A 16-day infection of Dictyocaulus viviparus in two groups of calves was treated with levamisole and fenbendazole respectively. Five days afterwards the calves were reinfected with 4000 larvae and necropsied 21 days later. Although the lungworm burdens of the two groups of calves were reduced by about 70 per cent compared to a control group the clinical signs of dyspnoea, tachypnoea and coughing were indistinguishable from a primary infection. This was due to pulmonary emphysema, oedema and an acute epithelialising pneumonia apparently associated with the death and disintegration of lungworms in situ, the result of an incompletely developed immune response. The results are compared with those obtained with the lungworm vaccine. It was concluded that the outcome of any system of "control" which depends on drug therapy and reinfection is unpredictable and that vaccination offers the only effective method of prophylaxis.

Some aspects of tick-borne diseases of British sheep.

The significance of tick-borne fever (TBF) and other tick-borne diseases of British sheep are reviewed. Experimental and field studies were carried out to clarify the role of TBF as a pathogen per se and as a predisposing factor in other diseases. Experimental TBF infection caused anorexia and depression in two- to three-week-old lambs, which under the stress of a hill environment could alone be a cause of mortality. Nine out of 10 lambs experimentally inoculated with Staphylococcus aureus during the febrile phase of a TBF reaction developed pyaemic lesions compared with four out of 20 lambs inoculated with S aureus alone. Specific pathogen-free lambs inoculated with an aerosol of Pasteurella haemolytica serotype A1 during a TBF reaction showed more severe clinical signs and had more extensive pathological changes at necropsy than control lambs given P haemolytica alone. Dual infection with TBF and louping-ill virus showed that not only were dually infected sheep more susceptible to louping-ill but almost all of them succumbed to a haemorrhagic syndrome involving a systemic mycotic infection with Rhizomucor pucillus. None of eight sheep given louping-ill virus alone developed this syndrome. Field studies indicated that morbidity and mortality in lambs in south-west Scotland could be markedly reduced by dipping and long acting antibiotic prophylaxis. Lamb groups in which both of these were carried out incurred losses of only 0.6 per cent compared with 10.3 per cent in control groups. In addition antibiotic-treated lamb groups demonstrated significantly better weight gains than untreated groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the epidemiology of bovine parasitic bronchitis.

In the West of Scotland the epidemiology of parasitic bronchitis in grazing calves was studied over a two year period with the aid of tracer calves and herbage examinations for Dictyocaulus viviparus larvae. The observations of both years emphasised the importance of overwintered lungworm larvae as a source of disease. In the first year it was shown that the ingestion and development of these overwintered larvae were, by themselves, directly responsible for severe morbidity, high faecal larval counts and deaths. In the second year it was shown that pasture ungrazed during the winter and spring and from which a hay crop was removed in mid-summer was still capable of producing clinical parasitic bronchitis in susceptible calves within three to four weeks of their introduction in later summer. In both years there was some evidence that the outbreaks appeared to be associated with the sudden availability of infective larvae on the herbage. The possibility that such larvae may have survived for many months in the soil is discussed. Despite the heavy challenge with lungworm larvae experienced by the grazing calves in the first year those vaccinated with lungworm vaccine survived, their clinical signs were mild and of short duration and their faecal larval output was greatly reduced.

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.