Trypanosoma congolense: Specific transformation in vitro of leukocytes from infected or immunized cattle

An assay to measure the specific proliferation in vitro of peripheral blood leukocytes (PBL) in response to ultrasonicated trypanosomes was adapted for use in cattle. The kinetics of mitosis exhibited by PBL from cattle which had been treated following infection with Trypanosoma congolense paralleled the development of a delayed-type skin reaction elicited with ultrasonicated and Formalin-fixed T. congolense. Responses in both tests were maximal on the fourth day after initiation. Specific proliferation of PBL harvested from cattle which had been immunized with intact, nonviable trypanosomes was also elicited in vitro by trypanosomal antigen. Peripheral blood leukocytes taken from cattle immunized with 50 μg of variant-specific surface antigen (VSSA) from T. brucei and from cattle infected with T. congolense were not stimulated to divide in vitro by ultrasonicated trypanosomes.     

Regulatory T cells enhance susceptibility to experimental trypanosoma congolense infection independent of mouse genetic background

Background

BALB/c mice are highly susceptible while C57BL/6 are relatively resistant to experimental Trypanosoma congolense infection. Although regulatory T cells (Tregs) have been shown to regulate the pathogenesis of experimental T. congolense infection, their exact role remains controversial. We wished to determine whether Tregs contribute to distinct phenotypic outcomes in BALB/c and C57BL/6 mice and if so how they operate with respect to control of parasitemia and production of disease-exacerbating proinflammatory cytokines.

Methodology/Findings

BALB/c and C57BL/6 mice were infected intraperitoneally (i.p) with 10³ T. congolense clone TC13 and both the kinetics of Tregs expansion and intracellular cytokine profiles in the spleens and livers were monitored directly ex vivo by flow cytometry. In some experiments, mice were injected with anti-CD25 mAb prior or post T. congolense infection or adoptively (by intravenous route) given highly enriched naïve CD25⁺ T lymphocytes prior to T. congolense infection and the inflammatory cytokine/chemokine levels and survival were monitored. In contrast to a transient and non significant increase in the percentages and absolute numbers of CD4⁺CD25⁺Foxp3⁺ T cells (Tregs) in C57BL/6 mouse spleens and livers, a significant increase in the percentage and absolute numbers of Tregs was observed in spleens of infected BALB/c mice. Ablation or increasing the number of CD25⁺ cells in the relatively resistant C57BL/6 mice by anti-CD25 mAb treatment or by adoptive transfer of CD25⁺ T cells, respectively, ameliorates or exacerbates parasitemia and production of proinflammatory cytokines.

Conclusion

Collectively, our results show that regulatory T cells contribute to susceptibility in experimental murine trypanosomiasis in both the highly susceptible BALB/c and relatively resistant C57BL/6 mice.     

Humoral antibody response and Ig characterization of the specific agglutinins in rabbits during experimental American trypanosomiasis

A comparative follow up study of the specific agglutinins detected by direct agglutination (DA) test and the immune response detected by specific lysis (SL), indirect immunofluorescence (IFA), indirect hemagglutination (IHA) and complement fixation (CF) tests in rabbits inoculated with trypomastigotes of T. cruzi is reported here.The specific antibody response was detected first by DA test. Reductive cleavage of sera with 2-mercaptoethanol produced a drop in the agglutinin titer of the sera during the first 30 days of infection.The next test to become positive was SL and later on the IFA, IHA and CF tests became positive simultaneously.When fractions obtained by column chromatography in Sephadex G-200 were tested serologically it was demonstrated that specific antibodies were detected mainly in fraction I (IgM) of the pooled rabbit sera obtained 15 days after inoculation (acute stage), and in fraction II (IgG) of the pooled sera obtained from rabbits 90 days after inoculation (chronic stage).Antigens prepared with trypsinized and formolized epimastigotes of three T. cruzi strains, belonging to each one of the different immunological groups described, worked similarly in the detection of specific agglutinin antibodies.Trypanosoma cruzi agglutinins were highly specific in their reaction with their homologous T. cruzi antigens as was proved by the low agglutinin titer obtained in sera from infected rabbits when, instead of T. cruzi epimastigotes, promastigotes of L. donovani were used as antigen, and by the incapacity of this parasite to absorb the T. cruzi agglutinins.     

Modeling the control of trypanosomiasis using trypanocides or insecticide-treated livestock

Background

In Uganda, Rhodesian sleeping sickness, caused by Trypanosoma brucei rhodesiense, and animal trypanosomiasis caused by T. vivax and T. congolense, are being controlled by treating cattle with trypanocides and/or insecticides. We used a mathematical model to identify treatment coverages required to break transmission when host populations consisted of various proportions of wild and domestic mammals, and reptiles.

Methodology/Principal Findings

An Ro model for trypanosomiasis was generalized to allow tsetse to feed off multiple host species. Assuming populations of cattle and humans only, pre-intervention Ro values for T. vivax, T. congolense, and T. brucei were 388, 64 and 3, respectively. Treating cattle with trypanocides reduced R ₀ for T. brucei to <1 if >65% of cattle were treated, vs 100% coverage necessary for T. vivax and T. congolense. The presence of wild mammalian hosts increased the coverage required and made control of T. vivax and T. congolense impossible. When tsetse fed only on cattle or humans, R ₀ for T. brucei was <1 if 20% of cattle were treated with insecticide, compared to 55% for T. congolense. If wild mammalian hosts were also present, control of the two species was impossible if proportions of non-human bloodmeals from cattle were <40% or <70%, respectively. R ₀ was <1 for T. vivax only when insecticide treatment led to reductions in the tsetse population. Under such circumstances R ₀<1 for T. brucei and T. congolense if cattle make up 30% and 55%, respectively of the non-human tsetse bloodmeals, as long as all cattle are treated with insecticide.

Conclusions/Significance

In settled areas of Uganda with few wild hosts, control of Rhodesian sleeping sickness is likely to be much more effectively controlled by treating cattle with insecticide than with trypanocides.     

Trypanosoma congolense: Natural and acquired resistance in the bovine

A total of 42 animals of various ages were infected with Trypanosoma congolense to investigate age resistance. Ten of eleven animals between 4 months and 1 year of age survived the infection without treatment. Two of eleven animals in the age range of 1 to 2 years also survived the infection whereas all 20 animals between 2 and 5 years of age died or needed treatment to survive. Young animals which needed no treatment to survive were refractive to challenge for at least 1 year after their last patent parasitemia. Older animals which required treatment to survive were also challenged at intervals after therapy. Three animals infected for 49 to 75 days before treatment were rechallenged 198 to 296 days later. Extensions in prepatent periods ranged from 5 to 13 days when compared to controls and the resulting infections were of a relapsing nature followed by self-cure. Effects of this disease on clinical parameters of previously infected animals were minimal. One animal infected for 196 days and rechallenged 501 days later had a prepatent period of 14 days as compared to 5 days for controls. This animal developed a brief relapsing infection followed by self-cure. Animals which were infected for periods of 41 to 77 days, received treatment, and were then rechallenged from 600 to 900 days later, showed some resistance to infection. Prepatent periods were extended from 1 to 3 days over those of control animals and although the resulting disease was severe, one of four animals self-cured without treatment. When animals which had self-cured primary challenges were rechallenged at periods up to 2 years later, they were completely refractory. When 12 animals which were presumed to be immune to syringe-passaged T. congolense were challenged by tsetse fly bite with the same strain of trypanosome, an appreciable immunity was evident. Five of twelve immune animals did not become patent while the other seven developed mild infections without severe clinical signs. All nine controls developed severe infections with eight requiring treatment to survive. When animals immune to the Trans-Mara I strain of T. congolense were challenged either by syringe or tsetse fly bite with a heterologous strain of T. congolense obtained from a different geographical area, no evidence of immunity was detected.     

Identification of a 4-fluorobenzyl l-valinate amide benzoxaborole (AN11736) as a potential development candidate for the treatment of Animal African Trypanosomiasis (AAT)

Novel l-valinate amide benzoxaboroles and analogues were designed and synthesized for a structure-activity-relationship (SAR) investigation to optimize the growth inhibitory activity against Trypanosoma congolense (T. congolense) and Trypanosoma vivax (T. vivax) parasites. The study identified 4-fluorobenzyl (1-hydroxy-7-methyl-1,3-dihydrobenzo[c][1,2]oxaborole-6-carbonyl)-l-valinate (5, AN11736), which showed IC₅₀ values of 0.15?nM against T. congolense and 1.3?nM against T. vivax, and demonstrated 100% efficacy with a single dose of 10?mg/kg against both T. congolense and T. vivax in mouse models of infection (IP dosing) and in the target animal, cattle, dosed intramuscularly. AN11736 has been advanced to early development studies.     

Development of Real Time PCR to Study Experimental Mixed Infections of T. congolense Savannah and T. b. brucei in Glossina morsitans morsitans

Tsetse flies are able to acquire mixed infections naturally or experimentally either simultaneously or sequentially. Traditionally, natural infection rates in tsetse flies are estimated by microscopic examination of different parts of the fly after dissection, together with the isolation of the parasite in vivo. However, until the advent of molecular techniques it was difficult to speciate trypanosomes infections and to quantify trypanosome numbers within tsetse flies. Although more expensive, qPCR allows the quantification of DNA and is less time consuming due to real time visualization and validation of the results. The current study evaluated the application of qPCR to quantify the infection load of tsetse flies with T. b. brucei and T. congolense savannah and to study the possibility of competition between the two species. The results revealed that the two qPCR reactions are of acceptable efficiency (99.1% and 95.6%, respectively), sensitivity and specificity and can be used for quantification of infection load with trypanosomes in experimentally infected Glossina morsitans morsitans. The mixed infection of laboratory Glossina species and quantification of the infection suggests the possibility that a form of competition exists between the isolates of T. b. brucei and T. congolense savannah that we used when they co-exist in the fly midgut.     

The B Cell Adaptor Molecule Bam32 Is Critically Important for Optimal Antibody Response and Resistance to Trypanosoma congolense Infection in Mice

Background

Bam32, a 32 kDa adaptor molecule, plays important role in B cell receptor signalling, T cell receptor signalling and antibody affinity maturation in germinal centres. Since antibodies against trypanosome variant surface glycoproteins (VSG) are critically important for control of parasitemia, we hypothesized that Bam32 deficient (Bam32-/-) mice would be susceptible to T. congolense infection.

Methodology/Principal Findings

We found that T. congolense-infected Bam32^-/- mice successfully control the first wave of parasitemia but then fail to control subsequent waves and ultimately succumb to their infection unlike wild type (WT) C57BL6 mice which are relatively resistant. Although infected Bam32^-/- mice had significantly higher hepatomegaly and splenomegaly, their serum AST and ALT levels were not different, suggesting that increased liver pathology may not be responsible for the increased susceptibility of Bam32^-/- mice to T. congolense. Using direct ex vivo flow cytometry and ELISA, we show that CD4+ T cells from infected Bam32^-/- mice produced significantly increased amounts of disease-exacerbating proinflammatory cytokines (including IFN-γ, TNF-α and IL-6). However, the percentages of regulatory T cells and IL-10-producing CD4+ cells were similar in infected WT and Bam32^-/- mice. While serum levels of parasite-specific IgM antibodies were normal, the levels of parasite-specific IgG, (particularly IgG1 and IgG2a) were significantly lower in Bam32^-/- mice throughout infection. This was associated with impaired germinal centre response in Bam32^-/- mice despite increased numbers of T follicular helper (Tfh) cells. Adoptive transfer studies indicate that intrinsic B cell defect was responsible for the enhanced susceptibility of Bam32^-/- mice to T. congolense infection.

Conclusions/Significance

Collectively, our data show that Bam32 is important for optimal anti-trypanosome IgG antibody response and suppression of disease-promoting proinflammatory cytokines and its deficiency leads to inability to control T. congolense infection in mice.     

Babesia microti: Pathogenesis of parasite of human origin in the hamster

The pathogenesis of the disease in hamsters caused by the first human Babesia isolant, tentatively named Babesia microti, and the immunologic relationship of the organism to Babesia canis were studied. The patent phase of the disease was characterized by severe anemia and marked parasitemia which occurred between the 6th and 41st day following infection. An increase in total white cell count with a neutrophilia, eosinophilia, monocytosis, and lymphocytosis was observed during the patent phase. The patent phase was followed by development of a carrier state. This was demonstrated by relapse following splenectomy 113 days after infection. No statistically significant differences were observed between the serum profiles of infected and noninfected animals during the period monitored. A serologic relationship between B. microti and B. canis was revealed by the use of gel diffusion and indirect fluorescent antibody (IFA) tests. The IFA test was used to monitor serum antibody responses during the patent and carrier phases of the disease. Crossabsorption studies between B. canis and B. microti revealed that the two organisms possess common and specific antigens.     

Recombinant expression and biochemical characterisation of two alanyl aminopeptidases of Trypanosoma congolense

Trypanosoma congolense is a haemoprotozoan parasite that causes African animal trypanosomosis, a wasting disease of cattle and small ruminants. Current control methods are unsatisfactory and no conventional vaccine exists due to antigenic variation. An anti-disease vaccine approach to control T. congolense has been proposed requiring the identification of parasitic factors that cause disease. Immunoprecipitation of T. congolense antigens using sera from infected trypanotolerant cattle allowed the identification of several immunogenic antigens including two M1 type aminopeptidases (APs). The two APs were cloned and expressed in Escherichia coli. As the APs were expressed as insoluble inclusion bodies it was necessary to develop a method for solubilisation and subsequent refolding to restore conformation and activity. The refolded APs both showed a distinct substrate preference for H-Ala-AMC, an optimum pH of 8.0, puromycin-sensitivity, inhibition by bestatin and amastatin, and cytoplasmic localisation. The two APs are expressed in procyclic metacyclic and bloodstream form parasites. Down-regulation of both APs by RNAi resulted in a slightly reduced growth rate in procyclic parasites in vitro.     

Trypanosoma congolense. I. Clinical observations of experimentally infected cattle

The course of disease was studied in 8 cattle infected with Trypanosoma congolense. Although the onset of patency was dependent on the numbers of infecting organisms, the duration of the infection was not. High fevers were present on the day of or the day after initial patency. Succeeding peaks of parasitemia, and a progressive weight loss of over 30% occurred. A decrease in packed cell volume (PCV) beginning the first week after infection was observed. Early in the course of the developing anemia, many polychromatophilic erythrocytes and occasional normoblasts were found in the blood. A leucopenia persisted for the duration of the disease. Total serum protein concentrations fell sharply during the first 5 weeks of infection, then gradually increased to low normal levels. Serum albumin levels followed a similar pattern for the first 5 weeks, and remained at a relatively low level. Although gamma globulin levels also declined during the first 5 weeks, their levels gradually surpassed those of preinfection samples. No marked changes in serum glucose were noted. A mild elevation of serum urea nitrogen values occurred early during infection, but subsided. The animals dying early after infection developed elevated total bilirubin levels.     

Diminazene Aceturate (Berenil) Modulates the Host Cellular and Inflammatory Responses to Trypanosoma congolense Infection

Background

Trypanosoma congolense are extracellular and intravascular blood parasites that cause debilitating acute or chronic disease in cattle and other domestic animals. Diminazene aceturate (Berenil) has been widely used as a chemotherapeutic agent for trypanosomiasis in livestock since 1955. As in livestock, treatment of infected highly susceptible BALB/c mice with Berenil leads to rapid control of parasitemia and survival from an otherwise lethal infection. The molecular and biochemical mechanisms of action of Berenil are still not very well defined and its effect on the host immune system has remained relatively unstudied. Here, we investigated whether Berenil has, in addition to its trypanolytic effect, a modulatory effect on the host immune response to Trypanosoma congolense.

Methodology/Principal Findings

BALB/c and C57BL/6 mice were infected intraperitoneally with T. congolense, treated with Berenil and the expression of CD25 and FoxP3 on splenic cells was assessed directly ex vivo. In addition, serum levels and spontaneous and LPS-induced production of pro-inflammatory cytokines by splenic and hepatic CD11b⁺ cells were determined by ELISA. Berenil treatment significantly reduced the percentages of CD25⁺ cells, a concomitant reduction in the percentage of regulatory (CD4⁺Foxp3⁺) T cells and a striking reduction in serum levels of disease exacerbating pro-inflammatory cytokines including IL-6, IL-12, TNF and IFN-γ. Furthermore, Berenil treatment significantly suppressed spontaneous and LPS-induced production of inflammatory cytokines by splenic and liver macrophages and significantly ameliorated LPS-induced septic shock and the associated cytokine storm.

Conclusions/Significance

Collectively, these results provide evidence that in addition to its direct trypanolytic effect, Berenil also modulates the host immune response to the parasite in a manner that dampen excessive immune activation and production of pathology-promoting pro-inflammatory cytokines, suggesting that this drug may also be beneficial for treatment of disease conditions caused by excessive production of inflammatory cytokines.     

A Novel qPCR Assay for the Detection of African Animal Trypanosomosis in Trypanotolerant and Trypanosusceptible Cattle Breeds

This study was conducted to (i) determine the prevalence of African Animal Trypanosomosis (AAT) in tsetse challenged areas, (ii) compare conventional with qPCR detection systems and (iii) evaluate the host genetic background and biology as risk factors. AAT prevalence studies are often confronted with low levels of parasitaemia. Hence, we designed a novel qPCR assay using primers and species specific probes amplifying the Internal Transcribed Spacer 1 (ITS1) gene. Thereby all three AAT species could be detected simultaneously. 368 individuals from three cattle types (Baoulé, Zebu and hybrids) originating from 72 farms in Burkina Faso were analysed. Farmers were interviewed and morphometric measurements of the cattle taken. A chi-squared test and a logistic regression model were calculated to detect associations with infection. In our study, the overall rate of prevalence detected with the novel qPCR assay was 11.14%. Compared to conventional PCR we identified a concordance of 91.30%. We tested 41 animals positive for trypanosome DNA, five animals showed multiple infections. Zebus were twice as often infected (21.74%) compared to Baoulé (9.70%) and hybrids (9.57%). Trypanosoma vivax is the dominant species (9.24%), as compared to T. congolense (2.44%) and T. brucei (0.82%). The chi-squared tests linking the infection events to the breeds (Zebu vs. Baoulé and Zebu vs. hybrids) were on the border of significance. No significant association with other tested parameters could be detected. We introduce a novel qPCR technique for the fast, sensitive and simultaneous detection of the three AAT species. Our results suggest that associations with breed and infection exist since Zebu cattle are more likely to be infected compared to Baoulé and hybrids. Indigenous taurine cattle breeds, like the Baoulé, therefore provide a unique and valuable genetic resource.     

Trypanosoma gambiense: immunity with thymic cell transfer in mice.

This report deals with the enhanced agglutinin production and protection in thymectomized, lethally irradiated mice (TI-mice) with transferred thymic cells from mice immune to T. gambiense. Such mice, when sensitized with trypanosome antigen showed protection against experimental infection and also produced agglutinins. Thymic cells from cortisone-treated immune mice were able to induce the production of agglutinins in TI-mice subsequently injected with antigen. However, these agglutinin titers were very low. In bovine serum albumin gradient centrifugation experiments, agglutinin production could be efficiently induced by inoculation of TI-mice with a rather high density thymic cell subpopulation taken from immune mice. Fractionated by Sephadex G-200, the agglutinins displayed a division into two parts, a first and second peak. The main agglutination reaction was seen in the first or macroglobulin peak. In the fractionation of serum by DEAE-cellulose column chromatography, agglutinins were eluted in two parts, the 0.0175 M and 0.4 M effluents. The agglutination by the 0.4 M effluent was much stronger than that of the 0.0175 M effluent, in agreement with the gel filtration results. The sera containing agglutinins were able to enhance the phagocytosis of trypanosomes by cultured macrophages from the peritoneal cavity of normal and irradiated mice. Delay of parasitemia was evident in some of the TI-mice having detectable agglutinins. The delayed parasitemia resulted from antigenically altered trypanosomes which were able to withstand the lethal factors of TI-mice. Transplantation of thymic cells was considered to be responsible for agglutinins induced by the antigenic stimulation in TI-mice and for protection against experimental infection.     

Effects of Trypanosoma congolense and nutritional supplements on establishment and outcome of pregnancy in trypanotolerant Djallonké ewes

Interactions between Trypanosoma congolense and nutritional status were studied in 42 ewes, bred at the peak of parasitaemia after synchronisation of oestrus. As experimental design a randomised block design was used with four treatment combinations (2×2 factors), of which two were on a restricted diet (L), the remainder on an unrestricted diet (H) and half of each nutritional group infected with T. congolense (LI, HI), the remainder serving as controls (LC, HC). Severity of parasitaemia was not influenced by supplementation and mortality rates were higher in the HI and LC groups, but these differences were found not significant. Progesterone levels during the synchronised cycle were significantly lower in the infected groups. Levels of pregnant specific protein B (PSPB) in pregnant sheep at days 21 and 26 were not significantly affected by nutrition or infection, despite the tendency of a decrease in infected groups. T. congolense clearly affected establishment of pregnancy, as shown by lower rates of pregnancy and extended intervals between breeding and confirmation of pregnancy, nor was there any benefit of nutritional supplementation. Mean progesterone concentration during pregnancy, in those ewes which lambed, was not different between groups. The effect of the T. congolense infection on the outcome of pregnancy was not clear with the LI and HC performing well and poor pregnancy outcomes in groups HI and LC, although differences in litter size might explain these anomalies. It is concluded that the most pronounced effect of T. congolense was a negative influence on establishment of pregnancy, with nutritional supplementation unable to overcome this effect but having a beneficial influence on maintenance and successful outcome of pregnancy. However, individual exceptions indicate that some ewes cope better with the negative effects of infection and poor nutrition.     

Precipitin responses of infected mice to exoantigens and cellular antigens of Trypanosoma musculi.

Plasma were collected from mice which had been immunosuppressed with 650 R from a cobalt-60 gamma radiation source and infected with Trypanosoma musculi. Trypanosomes were also collected from immuno-suppressed mice and from nonirradiated, infected animals. Rabbit antiserum was prepared against trypanosomes fron nonirradiated mice and employed in immunodiffusion analyses to detect trypanosome exoantigens (ExAg) in plasma of irradiated, infected mice and cellular antigens (CAg) in extracts of parasites which had been collected from immunosuppressed and nonirradiated hosts. The rabbit antiserum formed at least 3 precipitin lines with plasma from irradiated, infected mice and 8–9 precipitin lines with extracts of parasites which were obtained from immunosuppressed and untreated mice. Two of the precipitin reactions were against mouse plasma antigens (PAg). Lower levels of PAg appeared to be present in extracts of trypanosomes which were isolated from the irradiated mice than in those from nonirradiated animals.Mice synthesized antibodies against 1 ExAg which was demonstrable in immunodiffusion tests by 14 days after T. musculi infection. A single precipitin reaction was also seen after 21 days. One to 2 precipitin lines were formed with ExAg after 42 days of infection. Two to 3 precipitin lines formed between the ExAg and mouse antisera collected 98, 175 and 341 days after injection of the T. musculi.Similar immunodiffusion reactions were detected with CAg present in both the extracts of T. musculi which had been isolated from irradiated and those from nonirradiated mice and the mouse antisera. One to 2 precipitin lines were found between CAg and antisera from mice which had been infected for 14 days. Two precipitating antigen-antibody systems were seen with antisera collected after 21, 42 and 98 days and 2–3 precipitin reactions were formed between CAg and antisera collected from mice 175 and 341 days after infection.Absorption and immunodiffusion analyses conducted with rabbit and mouse antisera indicated parasite ExAg in plasma of irradiated, T. musculi infected mice were also present in preparations of CAg of the trypanosomes. The persistence of antibody and the increase in the numbers of antigen-antibody systems detected by immunodiffusion during the course of the infection may in part be related to the presence of parasites in capillaries of the kidneys long after they cannot be demonstrated in the peripheral blood of the host.     

Trypanosoma vivax GM6 Antigen: A Candidate Antigen for Diagnosis of African Animal Trypanosomosis in Cattle

Background

Diagnosis of African animal trypanosomosis is vital to controlling this severe disease which hampers development across 10 million km² of Africa endemic to tsetse flies. Diagnosis at the point of treatment is currently dependent on parasite detection which is unreliable, and on clinical signs, which are common to several other prevalent bovine diseases.

Methodology/Principle Findings

the repeat sequence of the GM6 antigen of Trypanosoma vivax (TvGM6), a flagellar-associated protein, was analysed from several isolates of T. vivax and found to be almost identical despite the fact that T. vivax is known to have high genetic variation. The TvGM6 repeat was recombinantly expressed in E. coli and purified. An indirect ELISA for bovine sera based on this antigen was developed. The TvGM6 indirect ELISA had a sensitivity of 91.4% (95% CI: 91.3 to 91.6) in the period following 10 days post experimental infection with T. vivax, which decreased ten-fold to 9.1% (95% CI: 7.3 to 10.9) one month post treatment. With field sera from cattle infected with T. vivax from two locations in East and West Africa, 91.5% (95% CI: 83.2 to 99.5) sensitivity and 91.3% (95% CI: 78.9 to 93.1) specificity was obtained for the TvGM6 ELISA using the whole trypanosome lysate ELISA as a reference. For heterologous T. congolense field infections, the TvGM6 ELISA had a sensitivity of 85.1% (95% CI: 76.8 to 94.4).

Conclusion/Significance

this study is the first to analyse the GM6 antigen of T. vivax and the first to test the GM6 antigen on a large collection of sera from experimentally and naturally infected cattle. This study demonstrates that the TvGM6 is an excellent candidate antigen for the development of a point-of-treatment test for diagnosis of T. vivax, and to a lesser extent T. congolense, African animal trypanosomosis in cattle.     

The relationship between the presence of antibodies and direct detection of Toxoplasma gondii in slaughtered calves and cattle in four European countries

In cattle, antibodies to Toxoplasma gondii infection are frequently detected, but evidence for the presence of T. gondii tissue cysts in cattle is limited. To study the concordance between the presence of anti-T. gondii IgG and viable tissue cysts of T. gondii in cattle, serum, liver and diaphragm samples of 167 veal calves and 235 adult cattle were collected in Italy, the Netherlands, Romania and the United Kingdom. Serum samples were tested for anti-T. gondii IgG by the modified agglutination test and p30 immunoblot. Samples from liver were analyzed by mouse bioassay and PCR after trypsin digestion. In addition, all diaphragms of cattle that had tested T. gondii-positive (either in bioassay, by PCR on trypsin-digested liver or serologically by MAT) and a selection of diaphragms from cattle that had tested negative were analyzed by magnetic capture quantitative PCR (MC-PCR). Overall, 13 animals were considered positive by a direct detection method: seven out of 151 (4.6%) by MC-PCR and six out of 385 (1.6%) by bioassay, indicating the presence of viable parasites. As cattle that tested positive in the bioassay tested negative by MC-PCR and vice-versa, these results demonstrate a lack of concordance between the presence of viable parasites in liver and the detection of T. gondii DNA in diaphragm. In addition, the probability to detect T. gondii parasites or DNA in seropositive and seronegative cattle was comparable, demonstrating that serological testing by MAT or p30 immunoblot does not provide information about the presence of T. gondii parasites or DNA in cattle and therefore is not a reliable indicator of the risk for consumers.     

Trypanosoma rhodesiense: variant specificity of immunity induced by irradiated parasites.

Rats immunized with irradiated Trypanosoma rhodesiense resisted infection with the homologous strain. When similarly immunized rats were challenged with parasites obtained from rhesus monkeys infected with the same strain, resistance depended on when parasites were obtained from the donor monkeys. Immunized rats challenged with trypanosomes obtained from a monkey during the first peak of parasitemia were solidly immune; immunized rats challenged with trypanosomes obtained from monkeys after their initial peak of parasitemia all succumbed to the challenging infection. These observations indicate that parasites of a variant antigenic specificity arose during the course of the monkey infections. Neutralization tests performed on the various isolates from rats and monkeys using antiserum obtained from immunized rats confirmed that the immunity produced by irradiated trypanosomes was variant specific.     

Theileria annulata: cross-reactions between a cell culture schizont antigen and antigens of East African species in the indirect fluorescent antibody test

A schizont antigen for the indirect fluorescent antibody test was prepared from an in vitro culture suspension of lymphoid cells infected with Theileria annulata macroschizonts. Two cattle recovered from T. annulata infection showed marked rises in antibody titer to this schizont antigen, with peak titers of 1:40,960 and 1:2560. Using sera from these recovered cattle, T. annulata cell culture schizont antigen was shown to cross-react markedly with Theileria parva and Theileria lawrencei cell culture schizonts and with Theileria mutans piroplasms in the indirect fluorescent antibody test. In contrast, using high-titer antisera to T. parva, T. lawrencei, and T. mutans, serological cross-reactions with T. annulata schizonts were only detected with T. parva and T. lawrencei antisera, and in both instances these were minimal. The value of the indirect fluorescent antibody test in the differential diagnosis of Theileria species pathogenic for cattle is discussed.