The response of pigs experimentally infected with trypanosoma brucei to isometamidium chloride therapy and the relation to nutrition

Summary

Growing pigs were placed on feeds with high (Group A), medium (B) and low (C) dietary energy and were infected with a virulent stock of T. brucei. Eight weeks later, the infected pigs were treated with isometamidium chloride at 1mg/kg live weight and all pigs were subsequently placed on a high energy diet to investigate their response to therapy.

Clearance of T. brucei from blood was completed 72h after treatment. There was no evidence of relapsed infection up to eight weeks after treatment. Red blood cell parameters returned to normal four to six weeks after treatment with responses being fastest in Group A, B and C had gained about two‐thirds of the live weight gains of their non‐infected pair‐fed controls.

It appears that the retarded weight gain as a result of the infection persisted after therapy since drug‐treated pigs did not gain as much weight as their non‐infected controls.     

Relapses in dogs experimentally infected with Trypanosoma brucei and treated with diminazene aceturate or isometamidium chloride

Twenty dogs of mixed local East African breeds were used. Five of the dogs were uninfected controls and 15 were infected with T. brucei (ILRAD 273). Five of the infected dogs were untreated controls, five were treated with a high curative dose of diminazene aceturate, (7 mg kg-1 body weight (wt.), and five were given a subcurative dose of isometamidium chloride (1 mg kg-1 body wt.). The drugs, given at 8 days post infection (d.p.i..), led to apparent recovery. The antibody titres, however, remained high in both groups and at 42-49 d.p.i. there was at least one relapse in each treatment group. Parasite populations from relapsed animals were more resistant to the drugs than the original infecting populations. The implications of these findings are discussed.     

Pathogenicity of Trypanosoma brucei brucei in experimentally infected pigs.

An experimental infection of 4-to 5-month old pigs with a stock of Trypanosoma brucei brucei resulted in a high parasitaemia, anorexia, pyrexia and a decline in the packed cell volume by one third. Nervous sign of circling and wobbling of the hind legs occurred in one of the pigs which at necropsy revealed a very severe meningo-encephalitis and the presence of trypanosomes in the brain. These results confirm that T. b. brucei might cause a severe disease in pigs.     

Erythrocyte response to Trypanosoma brucei in experimentally infected dogs.

Trypanosoma brucei infection produced an acute and fatal disease in Nigerian mongrel dogs due to a rapidly developing anaemia. Infected dogs responded with increased reticulocytosis, which was not sustained with chronicity. In comparison the response to artificially-induced haemolytic anaemia was progressive, marked and sustained. The anaemia of T. brucei infection of dogs was either normocytic normochromic in acute infection or microcytic normochromic in chronic infection. Artificially-induced haemolytic anaemia was either macrocytic normochromic or normocytic normochromic. The erythropoietic potential of plasma in vivo in mice increased in T. brucei-infected dogs except at the terminal parasitaemia. The anaemia in Trypanosoma brucei-infected dogs is therefore initially responsive but becomes poorly involved with chronicity.     

Relapse infection after chemotherapy in dogs experimentally infected with Trypanosoma brucei brucei

Studies on relapsing Trypanosoma brucei brucei infections in dogs after Berenil treatment revealed that the first relapse occurred 13 to 64 days after chemotherapy and 36 to 79 days after inoculation. A second relapse infection was observed in two dogs 43 and 60 days after a second Berenil treatment. During the aparasitaemic period following chemotherapy in four dogs, successful transmission (as evidenced by subsequent parasitaemia) following the intraperitoneal inoculation of homogenate of brain from two of the dogs into recipient rats was obtained. Transmission with blood collected just before the animals were sacrificed was, however, negative. Hornogenates of other organs (liver, spleen, eyes, testes, kidneys, heart and lymph node) were also non-infective. One dog inoculated with relapsed trypanosomes and treated with Berenil soon after showing parasitaemia was completely cured of the infection. It was considered that the brain is the source of relapse in T b brucei infection after Berenil therapy and that the relapse was not due to drug resistance.     

The blood volumes and erythrokinetics of Ndama and Zebu cattle experimentally infected with Trypanosoma brucei.

The responses of susceptible Ndama and Zebu cattle to experimental infection with Trypanosoma brucei were compared using haematological, parasitological and radioisotopic methods. Animals of both breeds became anaemic, but this was more severe in the Zebu cattle, one of which died. Although the prepatent period was the same in animals of both breeds, the levels of the first and subsequent peaks of parasitaemia were higher in the Zebu. The anaemia was due to an accelerated rate of red cell break-down which was more marked in the Zebu cattle. Haemodilution was not a feature. There was no evidence of dyshaemopoiesis but iron reutilisation from degraded erythrocytes was impaired. The greater resistance of the Ndama to T brucei infection could not be attributed to the capacity of this breed to mount a more effective erythropoietic response than the Zebu.     

Suppression of immune response to sheep red blood cells in rats experimentally infected with Trypanosoma brucei gambiense

A direct haemagglutination assay for antibodies to sheep red blood cells (SRBC) was used to assess the response of rats infected with Trypanosoma brucei gambiense. Whereas uninfected rats showed an efficient primary and secondary immune response to SRBC, trypanosome-infected rats displayed depressed antibody response starting about six days after infection. Infected rats failed to respond to a challenge dose of SRBC given 14 days after infection while uninfected control animals responded with an increased level of antibody production. These observations showed that T. b. gambiense infection inhibited both primary and secondary immune response to SRBC in rats. The result of this experiment is very important with regard to serological methods used to detect increasing levels of antibody production for diagnosis of diseases caused by bacterial and viral pathogens. In a concurrent trypanosome infection such increasing antibody levels would not be observed, leading to inaccurate diagnosis. Thus trypanosomiasis infection should be excluded under field conditions before the value of a serological diagnosis can be fully utilized.     

Spontaneous cure of domestic pigs experimentally infected by Trypanosoma brucei gambiense. Implications for the control of sleeping sickness

The existence of a pig reservoir for human African trypanosomosis (HAT) due to Trypanosoma brucei gambiense complicates the fight against this disease. This study, reports results obtained from pigs, which were inoculated with the blood of a person, suffering from HAT in Cameroon. The pigs were reared and kept in the shelter from all contact with Glossina, and monitored for 188 days. The seroconversion was checked by agglutination assays for trypanosomosis (CATT 1.3 and LATEX/T.b.gambiense). The parasitemia was measured by quantitative buffy coat method (QBC) and by polymerase chain reaction method (PCR). In addition, growth was recorded as well as blood counting and blood formulas. The results showed that the pigs were trypanotolerant and cure themselves in less than 6 months. It is concluded that sterilisation of this reservoir could be achieved by tsetse-control measures in 1 year. It confirms the strategy to complement screening and treatment of HAT with tsetse fly control measures.     

The pathogenesis of anaemia in goats experimentally infected with Trypanosoma congolense or Trypanosoma brucei: use of the myeloid:erythroid ratio

The present study examined the development of anaemia in Small East African goats experimentally infected with Trypanosoma congolense or Trypanosoma brucei. Experimental goats received a primary trypanosome challenge on day 0, treated with diminazene aceturate on day 49 and received a secondary trypanosome challenge on day 77 of the 136-day experiment. Both primary and secondary challenges were characterised by reduced peripheral erythrocyte counts, fall in packed cell volume (PCV), hypohaemoglobinaemia and reductions in the myeloid:erythroid ratios (M:E) compared with the uninfected goats. The progressive reduction in the M:E ratios denoted increased erythrogenesis in response to increased destruction of erythrocytes in blood by infecting trypanosomes or their products. The more rapid fall in M:E ratio in T. congolense infections shows that this parasite causes more severe clinical pathological effects in goats than T. brucei.     

Suggested dosage rates of melarsoprol in the treatment of mice experimentally infected with Trypanosoma brucei gambiense

One group of BALB/c mice infected with a highly virulent strain of Trypanosoma brucei gambiense were treated intraperitoneally with three series of three injections (each injection of 10 mg/kg) of Mel-B separated by seven days of rest, while a second group was treated once by a single injection All the Mel-B treated mice in both experiments were negative for parasites when examined using either the wet blood film or buffy coat methods, but were intermittently PCR positive during the sampling period. We encourage the use of a repeat negative PCR test over a one month period in combination with corroborative clinical and parasitological investigation to be suggestive of cure in experimental animals previously infected with trypanosomosis. In view of the exorbitant costs of Mel-B and its extreme toxicity, we recommend that Mel-B be given as one course of two injections (each equivalent to 10 mg/kg) separated by 2 d of rest in experimentally infected rodent models.     

The transmissibility of Trypanosoma congolense seems to be associated with its level of resistance to isometamidium chloride

In large parts of Africa the control of livestock trypanosomiasis relies on the use of trypanocidal drugs. Resistance against the available compounds is developing rapidly in the trypanosome population. The effect of the development of drug resistance on the fitness of the trypanosome is not well known. To determine the effect of the development of resistance to isometamidium chloride on the trypanosome's transmissibility, transmission experiments were conducted. Use was made of three isogenic clones of Trypanosoma congolense with different susceptibility to the drug. The infection rate in Glossina morsitans morsitans differed significantly between clones and was significantly higher in tsetse flies infected with the T. congolense clone with the highest level of drug resistance.     

The serological response of pigs experimentally infected with a species of Taenia from Taiwan

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibody in pigs infected with a possibly new species of Taenia isolated in Taiwan is described. The test antigen ThFAS was fractionated from the cyst fluod of a heterologous cestode Taenia hydatigena. In lightly infected pigs ( 4 recovered cysts at necropsy 17 weeks post-inoculation), antibody was detected as early as 3 weeks post-inoculation. In more heavily infected pigs (6–72 recovered cysts at necropsy 32 weeks post-inoculation), antibody was still detectable at the time of necropsy. Cysterci were found only in the livers of the infected pigs. This ELISA should be highly useful for detecting infection of pigs with this larval cestode in regions where the presence of Taenia solium is unlikely.     

Systemic antibody response in colostrum-deprived pigs experimentally infected with Haemophilus parasuis

The serum antibody response to an experimental infection by Haemophilus parasuis, the etiological agent of Glässer’s disease in pigs, was characterized by ELISA measuring IgM and IgGt levels against whole-cells and outer-membrane-proteins (OMPs) as antigens. Five groups of pigs were studied, four of those were previously immunized with different formulations, and the fifth was maintained as non-immunized control. All groups were challenged with 5 × 10⁹ CFU of H. parasuis. The non-commercial bacterin induced a full protection against disease, the OMP-vaccine and the exposure to a sublethal dose of 10⁵ CFU protected only partially, and the recombinant TbpB-vaccine conferred no protection. The humoral response in the pigs that died after infection (all controls, all those vaccinated with the recombinant TbpB, and two of both those inoculated with OMPs and those exposed to the sublethal dose) could be only measured before it, but it was irrelevant in all cases. However, a specific IgM and IgGt production was observed before challenge in all the surviving pigs, irrespective of the type of immunization received. This antibody response was even greater after H. parasuis infection, especially in those survivors receiving the sublethal dose. These results suggest a role of the antibodies developed after the different immunization protocols in preventing infection and death; therefore, the humoral immunity is protective against experimental Glässer’s disease.     

Absence of correlation between karyotype profiles of Trypanosoma congolense and resistance to isometamidium chloride

Chromosome profiles of 10 Trypanosoma (T.) congolense populations with different isometamidium sensitivities were compared using the pulsed field gel electrophoresis technique. The aim was to elucidate whether there was a karyotype pattern specific to eight isometamidium resistant phenotypes. Analysis of the profiles indicated that all populations displayed several discrete bands at the region of small, intermediate and large chromosomes. The highest similarity was observed between two isolates originating from Burkina Faso, indicating that they had the same genetic origin. Other eight strains exhibited different patterns in terms of chromosome size and numbers such that there was no characteristic karyotype pattern that was established specifically to identify resistant populations and discriminate them from the sensitive ones. This study has revealed that isometamidium resistance is not correlated to karyotype profile in T. congolense.     

Protective efficacy of isometamidium chloride and diminazene aceturate against natural Trypanosoma brucei, Trypanosoma congolense and Trypanosoma vivax infections in cattle under a suppressed tsetse population in Uganda

The protective efficacy of isometamidium chloride (ISMM) and diminazene aceturate (DIM) against Trypanosoma brucei, Trypanosoma congolense and Trypanosoma vivax infections in cattle under a suppressed tsetse population was assessed in southeast Uganda. A total of 66 and 57 trypanosome-infected cattle were treated with ISMM and DIM, respectively together with 177 trypanosome-free animals not treated were followed for 12 months, checked every 4 weeks. There was no statistical difference in the mean time to infection with any trypanosome species in animals treated with ISMM or DIM. However, the mean time to trypanosome infection was significantly longer for treated animals than controls. The mean time to infection with each of the three trypanosome species differed significantly, with the average time to T. vivax infection the lowest, followed by T. congolense and then T. brucei. The protective efficacy of DIM was as good as that of ISMM; implying curative treatments against trypanosomosis are sufficient for combination with tsetse control. Isometamidium chloride or DIM had the highest impact on T. brucei and T. congolense infections in cattle.     

Action of isometamidium chloride on the insect vector form of Trypanosoma vivax

The effect of isometamidium chloride upon developing and mature Trypanosoma vivax occurring in Glossina palpalis palpalis flies was evaluated. Newly emerged G p palpalis flies were infected with T vivax by allowing them to feed on parasitaemic animals. Two experiments were conducted and in each the flies were divided into two groups. One group of infected flies was fed in vitro through a membrane on defibrinated cow blood containing isometamidium chloride at 0.1 mg ml-1, after which they were dissected and examined for trypanosomes, and the other group was fed in the same way on unmedicated blood. The results showed that out of a total number of 129 flies which fed on medicated blood, none was infected, while 55 out of 127 flies which fed on unmedicated blood were infected. The results indicate that isometamidium chloride eliminated the insect vector form of T vivax. These findings are of potential significance in the control of trypanosomiasis in the field, particularly in the operation of the sterile insect technique.     

Parasitaemia and clinical manifestations in Trypanosoma brucei infected dogs.

Four dogs were infected with Trypanosoma brucei (Mkar strain) while another four were used as uninfected controls. Two of the dogs showed acute disease and died in the first wave of parasitaemia on days 7 and 8 post infection (PI) while the other two died from the sub-acute disease on days 24 and 28 PI corresponding to the second wave of parasitaemia. In the first wave of parasitaemia there was a sharp decrease in the packed cell volume, red blood cell, haemoglobin, total leucocytes, eosinophil, neutrophil and lymphocyte values, but during the period of low parasitaemia there was a slight recovery of the values of total leucocytes and lymphocytes although these and the other values showed a continuous decrease during the second wave of parasitaemia. In contrast, there was a consistent monocytosis in both acute and sub-acute diseases. The general picture was that of loss of condition, anaemia, leucopenia, monocytosis, ocular impairment, elevated temperature, pulse and respiratory rates, the difference between the acute and sub-acute diseases being in the degree of intensity. The degree of anaemia noted and the circulatory disturbances associated with the infection could have caused the death of all the infected dogs.     

Acute-phase protein response in pigs experimentally infected with Haemophilus parasuis

The acute-phase protein (APP) response to an infection caused by Haemophilus parasuis, the etiological agent of Glässer's disease in pigs, was characterized measuring serum concentrations of pig major acute-phase protein (pig MAP), haptoglobin (HPT), C-reactive protein (CRP) and apolipoprotein A-I (ApoA-I) in colostrum-deprived pigs. They were divided into six experimental groups: non-immunized control group (I); immunized with a non-commercial bacterin (II); with an OMP-vaccine (III); with a sublethal dose (IV); and with two commercial bacterins (V and VI). All groups were challenged intratracheally with 5 × 10⁹ CFU of H. parasuis 37 days after immunisation. The highest levels of the positive APPs (pig MAP, HPT and CRP) and the lowest levels of the negative APPs (ApoA-I) were observed in the animals that died as a consequence of the infection, both those in the non-inmunized and in the immunized groups. However, the surviving animals (all of them in groups II, V and VI, two pigs in group III, and three in group IV) showed a minor variation in APP response, mainly on day 1 post-challenge (p.c.), and then tended to recover the initial values. APP response was still less pronounced in the groups of pigs previously immunized with bacterins. In conclusion, APP response can reflect Glässer-disease ongoing, showing a correlation between the severity and duration of the clinical signs and lesions and the magnitude of changes in the APP levels.