Blood Incubation Infectivity Test Research Articles

The innate resistance of Trypanosoma copemani to human serum

Trypanosoma copemani is known to be infective to a variety of Australian marsupials. Characterisation of this parasite revealed the presence of stercorarian-like life-cycle stages in culture, which are similar to T. rangeli and T. cruzi. The blood incubation infectivity test (BIIT) was adapted and used to determine if T. copemani, like T. cruzi and T. rangeli, has the potential to grow in the presence of human serum. To eliminate any effects of anticoagulants on the complement system and on human high density lipoprotein (HDL), only fresh whole human blood was used. Trypanosoma copemani was observed by microscopy in all human blood cultures from day 5 to day 19 post inoculation (PI). The mechanism for normal human serum (NHS) resistance in T. copemani is not known. The results of this study show that at least one native Australian trypanosome species may have the potential to be infective for humans.

Blood incubation infectivity test (BIIT): usefulness as compared to the SRAPCR in the identification of Trypanosoma brucei rhodesiense in animal reservoirs

This investigation is aimed at comparing the Blood Incubation Infectivity Test (BIIT) with the Serum Resistance Associated gene Polymerase Chain Reaction (SRA-PCR) techniques as tools that can be used to detect the presence of Trypanosoma brucei rhodesiense parasites in domestic animals reservoirs. Blood samples from 4754 animals (cattle, pigs and small ruminants) were screened for the presence of Trypanozoon parasites by microscopy and in total, 109 samples (98 T. brucei sl isolates from domestic animal and 11 known T. b. rhodesiense) were analysed. There was 81.6% agreement between the results from the BIIT and the SRAPCR, with Cohen Kappa of 0.63 suggesting a substantial agreement between the two tests. When SRA-PCR was taken as the reference standard, the BIIT test had a sensitivity of 82.1% and specificity of 81.4%. The positive and negative predictive values were 74.4% and 87.3% respectively. Therefore, although the BIIT still remains a feasible test for detecting human infective trypanosomes in many African laboratories, SRA-PCR offers a quicker method for the identification of the animal reservoir for T. b. rhodesiense to enable Governments make firm decisions on public good investment on treating infected animals for the purpose of controlling the zoonotic sleeping sickness.

Isolation of a human serum-resistant Trypanosoma brucei from a naturally infected pig in the Nsukka area of Enugu State

A strain of trypanosome was isolated from one of the trade pigs held at the Orie Orba market lairage in Udenu Local Government Area of Enugu State. It was identified by its motility in wet blood film and morphological characteristics in Giemsa-stained thin blood film as Trypanosoma brucei. To further characterize the parasite and identify to which of the brucei-type sub-species it belonged, it was subjected to the blood incubation infectivity test. Trypanosomes were incubated in phosphate-buffered saline glucose (PSG), normal pig serum or normal human serum for 4 hours at 37˚ C before aliquots of 200 μl containing 106 trypanosomes were used to infect each mouse in three groups of 10 mice per group. The animals were monitored daily for parasitaemia for a period of 30 days using routine parasitological techniques. All mice infected with parasites incubated in PSG and pig serum became parasitaemic with a mean pre-patent period of 3 and 8 days respectively. In contrast, only one of the 10 mice infected with trypanosomes incubated in human serum did not become parasitaemic, indicating that the strain is resistant to human serum and potentially infective to humans. It was concluded that this isolate is possibly Trypanosoma brucei gambiense and that this study supports the epidemiological claim that domestic pigs may serve as reservoir hosts for humansleeping sickness due to T. b. gambiense. Key Words: Trypanosoma brucei, Human serum resistance, Pigs, Reservoir host, T. b. gambiense (Nigerian Veterinary Journal: 2003 24(1): 37-43)

Natural immunity to human African trypanosomiasis: trypanosome lytic factors and the blood incubation infectivity test

This review focuses on the epidemiology of human African trypanosomiasis: why it occurs in humans, the current methods of surveillance, and the drugs available to treat it. Emphasis is placed on the identification of human-infective trypanosomes by the blood incubation infectivity test. This test distinguishes between trypanosomes that are non-infective for humans and those that are potentially infective. Currently the test requires incubation of parasites with human serum before injection into mice; any surviving parasites are considered human-infective. The factors in serum that kill all nonhuman-infective parasites are known as trypanosome lytic factors. The paper details the biochemistry of these factors and recommends standardization of the test based on current knowledge. This test can be used to screen animals with trypanosomiasis, in order to evaluate their role during endemic and epidemic human African trypanosomiasis.

Porcine cerebralTrypanosoma brucei brucei trypanosomiasis

This paper describes the investigation of a disease outbreak among 10 adult pigs in Nsukka, Anambra State, Nigeria. Prior to the investigation one sow died of the disease. Trypanosomes were later detected in the blood of two of the nine pigs subsequently investigated. All the pigs were then treated with deep intramuscular injection of 8 mg/kg diminazene aceturate (Berenil). Thirty six days after treatment a boar and a sow relapsed with signs similar to the ones shown previously. Further examination of their blood and faeces revealed nothing of parasitological significance. Following deteriorating condition and development of nervous signs the boar was salvaged while the sow died of the infection. Brain impression smears taken from both animals during postmortem examination revealed numerous trypanosome parasites identified by morphology and blood incubation infectivity test (BIIT) as Trypanosoma brucei brucei. The clinical and economic significance of the outbreak are discussed.

Occurrence of human serum-resistant Trypanosoma congolense in goats and sheep in Nigeria

An assessment of the role of dogs, goats and sheep as reservoir hosts of African trypanosomes infective for humans (sleeping sickness) was carried out in Nigeria during a 2-year study period. Twelve stocks of Trypanosoma (Trypanozoon) brucei, 10 stocks of Trypanosoma congolense and 11 stocks of Trypanosoma vivax were isolated from a total of 699 animals, comprising 286 sheep, 221 goats and 192 dogs. The potential infectivity of the isolates for man was tested in vitro using the blood incubation infectivity test. None of the T. brucei group was resistant to the trypanocidal action of human serum; three of the T. congolense group were resistant to human serum. A parallel study of the trypanocidal action of test serum on authenticated T. brucei brucei and T. brucei gambiense showed that the human serum behaved as expected. The possibility is discussed that T. congolense might produce infections in man and should, therefore, be handled carefully both in the laboratory and by veterinarians in the field.

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.

Variation in the sensitivity of successive variable antigen types in a Trypanosoma (Trypanozoon) brucei subspecies clone to some African game animal sera.

A Trypanosoma brucei subspecies clone was passaged in rats at ten-day intervals and the sensitivity/resistance to a variety of mammalian sera, of the successive variable antigen types (VATs) produced, was examined sequentially in a modified version of the blood incubation infectivity test (BIIT). A proven homogeneous VAT was used to initiate this series of tests, in which seven successive and different VATs were each exposed in vitro at 38.5 degrees C for 2 h to standard samples of pooled rat serum (PRS), normal human serum (NHS) and to sera from two different eland and three different hippopotami. Samples were then inoculated into susceptible rats to determine the effects of the individual sera on the subsequent infectivity of the trypanosomes. The seven VATs were found to vary widely in their sensitivity to the different game sera, though all remained strongly resistant to the pooled rat and the normal human serum samples. BII testing of isolates from positive test rats in the sequential study showed that their resistance to normal human serum was unaffected by their exposure in vitro to the different game sera.

Sleeping sickness survey in Musoma district, Tanzania: Further study on the vector role of Glossina

In a follow-up survey of sleeping sickness in the Ikoma-Serengeti region of Tanzania, carried out in May and June 1972, 11,060 G. swynnertoni and 95 G. pallidipes were collected, triturated mainly in batches of 50, and the supernate of each batch was inoculated into cyclophosphamide-treated mice. Nine strains of brucei-subgroup were isolated from G. swynnertoni, which indicated the vector rô1e of the local tsetses in the transmission cycle of infection in this endemic region. All the 9 brucei-subgroup isolates gave consistently negative results by the Blood Incubation Infectivity Test (BIIT) whereas the respective controls gave positive results. However, T. (T.) brucei and particularly T. (T.) rhodesiense standards which were tested simultaneously with the test strains gave inconsistent results. It would seem therefore that the BII test in its present form is not reliable for differentiating between the above two trypanosome species, and so the 9 brucei-subgroup isolates could not be classified further. In the Ikoma-Serengeti region there are no restricted foci of Rhodesian sleeping sickness but rather the various components comprising zoonosis interact sporadically throughout this part of Tanzania. The biocenosis of infection favours intense circulation of the parasites, mainly via the tsetse vectors, among game animals some of which function as natural reservoirs; in this ecological zone man is only an incidental host.

Blood incubation infectivity test results for Trypanosoma brucei sub-group isolates tested in the Lambwe Valley, South Nyanza, Kenya.

A total of167 Trypanosoma brucei sub-group strains, isolated from non-human hosts, were tested by the Blood Incubation Infectivity Test.

Trypanosoma brucei infection in a lion (panthera leo) in zambia

1.Trypanosoma brucei was isolated from a lion by means of mouse inoculation. 2. The strain was identified asT. brucei by the Blood Incubation Infectivity Test (BIIT). 3. BIIT was further simplified by using serum instead of plasma. 4. For effective working of BIIT the incubation of samples was shown to be essential.

The blood incubation infectivity test as a means of distinguishing between Trypanosoma brucei brucei and T. brucei rhodesiense

Targett G. A. T. and Wilson V. C. L. C. 1973. The blood incubation infectivity test as a means of distinguishing between Trypanosoma brucei brucei and T. brucei rhodesiense. International Journal for Parasitology, 3: 5–11. A simple test for distinguishing between the morphologically identical subspecies Trypanosoma brucei rhodesiense, which is infective to man, and T. brucei brucei, which by definition is not, has been described. This test, the blood incubation infectivity test (BIIT), is based on absolute differences in the infectivity to rats of the subspecies after exposure to human blood, and was applied to strains which are preserved in the laboratory as stabilates. Five T. brucei brucei strains were BIIT negative since their infectivity was destroyed by incubation in normal human blood but only five of the nine T. brucei rhodesiense strains tested were consistently BIIT positive. The other four gave equivocal results, indicating that the resistance of T. brucei rhodesiense strains to the trypanocidal effect of human blood can change, probably as a result of maintenance in the laboratory.

The differentiation of Trypanosoma rhodesiense from T. brucei by means of human serum

1. 1. A review is made of the Blood Incubation Infectivity Test (BIIT) introduced by Rickman and Robson (1970) to differentiate Trypanosoma rhodesiense from T. brucei in the laboratory. This test consists of incubating the trypanosomes with human blood and then injecting them into a rat; if the rat becomes infected, the trypanosomes are probably T. rhodesiense, and conversely. 2. 2. In the present paper it is shown that most of the effect of the human blood or serum is exerted after injection into the animal, and not during the previous incubation in vitro; moreover, the effect is proportional to the dose of blood per weight of animal. Accordingly, improved methods of conducting the test are recommended, especially the inoculation of the trypanosomes—blood mixture into 5 mice (rather than 1 rat), each animal to receive at least 0·4 ml. human blood per 20 g. weight. 3. 3. Further, the test should be interpreted according to statistical definitions of what is to be accepted as “serum-resistance”. Provisionally a strain which can infect 20% of the animals (treated with human blood as above) might well be regarded as “serum-resistant” (probably T. rhodesiense) and so potentially dangerous to man.

The repeatability of the blood incubation infectivity test

The repeatability of the blood incubation infectivity test

The trypanocidal action of human serum and of baboon plasma

1. 1. Investigations have been made on the activity of normal human serum, and of baboon plasma in killing non-pathogenic trypanosomes such as Trypanosoma equiperdum. Baboon plasma is 5 times more active than human serum but otherwise its activity is qualitatively the same. 2. 2. The activity varies according to whether it is measured in vitro or in vivo (i.e. in mice). It is postulated that there are 2 closely related substances, A which acts in vitro and B which acts in vivo. Usually both substances are present but in suitable circumstances they can be obtained separately. Most human and baboon sera contain much B, but some contain very little A. Usually there is 20–40 times as much B as A. 3. 3. In vitro, there is a lag period of about 3 4 hour and then trypanocidal action is completed in less than 6 hours. In vivo the action is a little slower. 4. 4. Substance A is suppressed if the human serum is injected intravenously into rats and mice but B is made more active. The substances could not be removed from serum by absorption with large numbers of trypanosomes. 5. 5. Human serum and baboon plasma were fractionated by ion-exchange chromatography and by gel filtration using Sephadex G-150 and Sepharose 6B. The active fractions were further examined by Laurell immunoelectrophoresis. These techniques indicated that the active substances are α 2 macroglobulins with molecular weight approximately 300,000. B has a slightly greater molecular weight and is slightly more acidic than A. It is postulated that both substances possess the same protein nucleus, but that they differ slightly in the configuration and/or composition. Some sera, which were initially inactive in vitro, yielded highly active fractions after gel filtration; this activation might be due to conversion of B into A or to removal of inhibitors which were masking A. 6. 6. When trypanosomes are exposed to serum there is a latent period of half an hour, after which the trypanosomes become distorted until they are almost globular. As shown by the electron microscope, there is progressive disintegration of the cytoplasm and large clear spaces appear beneath the pellicle. The other structures of the trypanosome persist unchanged for a long period, but ultimately the trypanosome disintegrates. 7. 7. As shown above the trypanocidal substances are α 2 macroglobulins and they are probably synthesized in the liver (not in the spleen and lymph glands). Accordingly their activity is quite different from the standard immunological processes. Their action might be due to either a coincidental stimulation of the serum protein by the surface of the trypanosome, or it might be a biological mechanism to protect men and baboons from many species of African trypanosomes, or that it might be part of a properdin system against infections in general. 8. 8. The trypanocidal action of human serum has been used by Rickman and Robson (1970) in the Blood Incubation Infectivity Test to differentiate T. rhodesiense from T. brucei. Improved methods for conducting and interpreting this test will be described in a second communication.

The use of human plasma in the blood incubation infectivity test to differentiate trypanosoma brucei and trypanosoma rhodesiense

The infectivity test of Rickman and Robson was shown to be an excellent method for distinguishing betweenT. brucei andT. rhodesiense in the small number of samples tested. A modification of the test by the use of human plasma in lieu of whole blood revealed that a one-half hour incubation was long enough for the factor found present in human plasma to act as an inhibitor of infection of mice withT. brucei.