Antigen detection ELISA: A sensitive and reliable tool for the detection of active infection of surra.

Trypanosomosis, an endemic disease in Asia, America (central and south) and Africa causes havoc economical loss in livestock industry. The carrier animals which are symptomless and harbours low level of parasites can act as a source of infection. The level of parasitaemia fluctuates, especially during the latent infection; moreover the antibodies which are not found early in the infection may persit even after recovery or chemotherapy. The parasitological and/or serological tests always can not detect current infection or carrier animals. Hence, in the present study double antibody sandwitch antigen detection ELISA (Ag-ELISA) is developed to detect circulating trypanosomes. The new assay has been evaluated using 554 field samples comprising bovine and camel. The diagnostic sensitivity and specificity of the new assay was found to be 97.4% and 99.0% respectively, with a Cohen's kappa value of 0.96. The developed assay could detect 11.5 Trypanosoma evansi per mL from the experimentally infected blood, buffy coat and purified T. evansi samples. The findings revealed that the developed assay can be exploited as a potential diagnostic tool in the detection of active trypanosomal infection.

Serum biochemical parameters and cytokine profiles associated with natural African trypanosome infections in cattle.

BACKGROUND: Animal African trypanosomiasis (AAT) greatly affects livestock production in sub-Saharan Africa. In Ghana prevalence of AAT is estimated to range between 5 and 50%. Studies have reported serum biochemical aberrations and variability in cytokine profiles in animals during infection. However, information regarding the biochemical parameters and cytokine profiles associated with natural infections are limited. This study was therefore aimed at investigating changes in the levels of serum biochemical parameters and inflammatory cytokines during a natural infection. METHODS: Nested internal transcribed spacer (ITS)-based PCR and sequencing were used to characterise trypanosome infection in cattle at two areas in Ghana (Adidome and Accra) of different endemicities. The cattle were sampled at four to five-week intervals over a period of six months. Levels of serum biochemical parameters, including creatinine, cholesterol, alkaline phosphatase (ALP), alanine aminotransferase (ALT), total bilirubin and total protein and cytokines (interleukin 10, interleukin 4, interleukin 12, interferon gamma and tumor necrosis factor alpha) were measured in serum samples and then compared between infected cattle and uninfected controls. RESULTS: The predominant trypanosome species detected in Accra (non-endemic) and Adidome (endemic) were Trypanosoma theileri and Trypanosoma vivax, respectively. Serum biochemical parameters were similar between infected and uninfected cattle in Accra. Infected cattle at Adidome however, had significantly higher levels of ALP, creatinine, total protein and total bilirubin (P < 0.05) and significantly lower levels of cholesterol (P < 0.05) at specific time points. At basal levels and during infection, significantly higher pro-inflammatory to anti-inflammatory (Th1/Th2) cytokine ratios were observed in cattle at Adidome compared to Accra (P < 0.05), indicating a shift towards Th1 immune response in Adidome. Levels of IL-10 were, however, significantly elevated in infected cattle in Accra (P < 0.05), suggesting high anti-inflammatory cytokine response in Accra. CONCLUSION: These results suggests that cattle in an endemic area repeatedly infected with trypanosomes of different species or different antigenic types demonstrate high pro-inflammatory (Th1) immune response and biochemical alterations whereas cattle in a non-endemic area with predominantly chronic T. theileri infections demonstrate high anti-inflammatory response and no biochemical alterations.

Molecular identification of trypanosome species in trypanotolerant cattle from the south of Gabon.

The aim of this study was to provide information on trypanosome species infecting trypanotolerant cattle from southern Gabon. The study was conducted on 224 trypanotolerant cattle from three regions located in southern Gabon, using ITS1 primer-based PCR. Seventy-two (32%) N'dama cattle were found polymerase chain reaction (PCR) positive with trypanosomes. The overall prevalence of trypanosomosis was 57% (63/110), 4% (4/100), and 36% (5/14) in the Gala section of the Nyanga ranch, the Miyama ranch, and Ossiele, respectively. Trypanosoma congolense and Trypanosoma vivax were identified. In Gala section and Ossiele, T. congolense and T. vivax were found. In the Miyama ranch, only T. vivax was identified. Mixed infections were also found. The forest (9%) and savannah (63%) subgroups of T. congolense were identified. The presence of the two subgroups was detected in 16 out of 56 cattle (29%). T. congolense and T. vivax would appear to be the main agents responsible for bovine trypanosomosis in southern Gabon. Although trypanotolerant, N'dama cattle may serve as a reservoir, and this should be further studied. On the other hand, these trypanotolerant cattle can be reared in such tsetse infested areas, which gives them an advantage compared to other trypanosensitive breeds, and this shows that they represent a key factor in biodiversity which has to be promoted.

Characterization of Calflagin, a Flagellar Calcium-Binding Protein from Trypanosoma congolense.

BACKGROUND: Identification of species-specific trypanosome molecules is important for laboratory- and field-based research into epidemiology and disease diagnosis. Although Trypanosoma congolense is the most important trypanosome pathogen of cattle in Africa, no species-specific molecules found in infective bloodstream forms (BSF) of the parasites have been identified, thus limiting development of diagnostic tests. METHODS: Immuno-mass spectrometric methods were used to identify a protein that is recognized by a T. congolense-specific monoclonal antibody (mAb) Tc6/42.6.4. The identified molecule was expressed as a recombinant protein in E. coli and was tested in several immunoassays for its ability to interact with the mAb. The three dimensional structure of the protein was modeled and compared to crystal- and NMR-structures of the homologous proteins from T. cruzi and T. brucei respectively, in order to examine structural differences leading to the different immunoreactivity of the T. congolense molecule. Enzyme-linked immunosorbent assays (ELISA) were used to measure antibodies produced by trypanosome-infected African cattle in order to assess the potential for use of T. congolense calflagin in a serodiagnostic assay. RESULTS: The antigen recognized by the T. congolense-specific mAb Tc6/42.6.4 was identified as a flagellar calcium-binding protein, calflagin. The recombinant molecule showed immunoreactivity with the T. congolense-specific mAb confirming that it is the cognate antigen. Immunofluorescence experiments revealed that Ca2+ modulated the localization of the calflagin molecule in trypanosomes. Structural modelling and comparison with calflagin homologues from other trypanosomatids revealed four non-conserved regions on the surface of the T. congolense molecule that due to differences in surface chemistry and structural topography may form species-specific epitopes. ELISAs using the recombinant calflagin as antigen to detect antibodies in trypanosome-infected cattle showed that the majority of cattle had antibody responses. Area under the Receiver-Operating Characteristic (ROC) curves, associated with host IgG and IgM, were calculated to be 0.623 and 0.709 respectively, indicating a positive correlation between trypanosome infection and the presence of anti-calflagin antibodies. CONCLUSIONS: While calflagin is conserved among different species of African trypanosomes, our results show that T. congolense calflagin possesses unique epitopes that differentiate this protein from homologues in other trypanosome species. MAb Tc6/42.6.4 has clear utility as a laboratory tool for identifying T. congolense. T. congolense calflagin has potential as a serodiagnostic antigen and should be explored further for its utility in antigen-detection assays for diagnosis of cattle infections.

Lack of specific alleles for the bovine chemokine (C-X-C) receptor type 4 (CXCR4) gene in West African cattle questions its role as a candidate for trypanotolerance.

A panel of 81 Asian, African and European cattle (Bos taurus and B. indicus) was analysed for the whole sequence of the CXCR4 gene (3844bp), a strong candidate for cattle trypanotolerance. Thirty-one polymorphic sites identified gave 31 different haplotypes. Neutrality tests rejected the hypothesis of either positive or purifying selection. Bayesian phylogenetic tree showed differentiation of haplotypes into two clades gathering genetic variability predating domestication. Related with clades definition, linkage disequilibrium analyses suggested the existence of one only linkage block on the CXCR4 gene. Two tag SNPs identified on exon 2 captured 50% of variability. Whatever the analysis carried out, no clear separation between cattle groups was identified. Most haplotypes identified in West African taurine cattle were also found in European cattle and in Asian and West African zebu. West African taurine samples did not carry unique variants on the CXCR4 gene sequence. The current analysis failed in identifying a causal mutation on the CXCR4 gene underlying a previously reported QTL for cattle trypanotolerance on BTA2.

Immunogenicity and Serological Cross-Reactivity of Saliva Proteins among Different Tsetse Species.

Tsetse are vectors of pathogenic trypanosomes, agents of human and animal trypanosomiasis in Africa. Components of tsetse saliva (sialome) are introduced into the mammalian host bite site during the blood feeding process and are important for tsetse's ability to feed efficiently, but can also influence disease transmission and serve as biomarkers for host exposure. We compared the sialome components from four tsetse species in two subgenera: subgenus Morsitans: Glossina morsitans morsitans (Gmm) and Glossina pallidipes (Gpd), and subgenus Palpalis: Glossina palpalis gambiensis (Gpg) and Glossina fuscipes fuscipes (Gff), and evaluated their immunogenicity and serological cross reactivity by an immunoblot approach utilizing antibodies from experimental mice challenged with uninfected flies. The protein and immune profiles of sialome components varied with fly species in the same subgenus displaying greater similarity and cross reactivity. Sera obtained from cattle from disease endemic areas of Africa displayed an immunogenicity profile reflective of tsetse species distribution. We analyzed the sialome fractions of Gmm by LC-MS/MS, and identified TAg5, Tsal1/Tsal2, and Sgp3 as major immunogenic proteins, and the 5'-nucleotidase family as well as four members of the Adenosine Deaminase Growth Factor (ADGF) family as the major non-immunogenic proteins. Within the ADGF family, we identified four closely related proteins (TSGF-1, TSGF-2, ADGF-3 and ADGF-4), all of which are expressed in tsetse salivary glands. We describe the tsetse species-specific expression profiles and genomic localization of these proteins. Using a passive-immunity approach, we evaluated the effects of rec-TSGF (TSGF-1 and TSGF-2) polyclonal antibodies on tsetse fitness parameters. Limited exposure of tsetse to mice with circulating anti-TSGF antibodies resulted in a slight detriment to their blood feeding ability as reflected by compromised digestion, lower weight gain and less total lipid reserves although these results were not statistically significant. Long-term exposure studies of tsetse flies to antibodies corresponding to the ADGF family of proteins are warranted to evaluate the role of this conserved family in fly biology.

Expression of regulatory dendritic cell-related cytokines in cattle experimentally infected with Trypanosoma evansi.

Trypanosoma evansi causes wasting disease in many livestock. T. evansi infection gives rise to inflammatory immune responses, which contribute to the development of inflammation-associated tissue injury. We previously reported that regulatory dendritic cells (DCs), which act as potential regulators of inflammation, were activated in infected mice and transfer of regulatory DCs to infected mice prolonged their survival. However, the kinetics of regulatory DCs in cattle, which are natural hosts of T. evansi, remained unclear. In this study, we report that the expressions of CCL8 and IL-10, which promote the development of regulatory DCs, were up-regulated in cattle experimentally infected with T. evansi. This finding is potentially useful for studying the control strategy of T. evansi infection in cattle.

Variant surface glycoproteins from Venezuelan trypanosome isolates are recognized by sera from animals infected with either Trypanosoma evansi or Trypanosoma vivax.

Salivarian trypanosomes sequentially express only one variant surface glycoprotein (VSG) on their cell surface from a large repertoire of VSG genes. Seven cryopreserved animal trypanosome isolates known as TeAp-ElFrio01, TEVA1 (or TeAp-N/D1), TeGu-N/D1, TeAp-Mantecal01, TeGu-TerecayTrino, TeGu-Terecay03 and TeGu-Terecay323, which had been isolated from different hosts identified in several geographical areas of Venezuela were expanded using adult albino rats. Soluble forms of predominant VSGs expressed during the early infection stages were purified and corresponded to concanavalin A-binding proteins with molecular masses of 48-67 kDa by sodium dodecyl sulfate-polyacrylamide gel electropohoresis, and pI values between 6.1 and 7.5. The biochemical characterization of all purified soluble VSGs revealed that they were dimers in their native form and represented different gene products. Sequencing of some of these proteins yielded peptides homologous to VSGs from Trypanosoma (Trypanozoon) brucei and Trypanosoma (Trypanozoon) evansi and established that they most likely are mosaics generated by homologous recombination. Western blot analysis showed that all purified VSGs were cross-reacting antigens that were recognized by sera from animals infected with either T. evansi or Trypanosoma (Dutonella) vivax. The VSG glycosyl-phosphatidylinositol cross-reacting determinant epitope was only partially responsible for the cross-reactivity of the purified proteins, and antibodies appeared to recognize cross-reacting conformational epitopes from the various soluble VSGs. ELISA experiments were performed using infected bovine sera collected from cattle in a Venezuelan trypanosome-endemic area. In particular, soluble VSGs from two trypanosome isolates, TeGu-N/D1 and TeGu-TeracayTrino, were recognized by 93.38% and 73.55% of naturally T. vivax-infected bovine sera, respectively. However, approximately 70% of the sera samples did not recognize all seven purified proteins. Hence, the use of a combination of various VSGs for the diagnosis of animal trypanosomosis is recommended.

Association studies in QTL regions linked to bovine trypanotolerance in a West African crossbred population.

African animal trypanosomosis is a parasitic blood disease transmitted by tsetse flies and is widespread in sub-Saharan Africa. West African taurine breeds have the ability, known as trypanotolerance, to limit parasitaemia and anaemia and remain productive in enzootic areas. Several quantitative trait loci (QTL) underlying traits related to trypanotolerance have been identified in an experimentally infected F(2) population resulting from a cross between taurine and zebu cattle. Although this information is highly valuable, the QTL remain to be confirmed in populations subjected to natural conditions of infection, and the corresponding regions need to be refined. In our study, 360 West African cattle were phenotyped for the packed cell volume control under natural conditions of infection in south-western Burkina Faso. Phenotypes were assessed by analysing data from previous cattle monitored over 2 years in an area enzootic for trypanosomosis. We further genotyped for 64 microsatellite markers mapping within four previously reported QTL on BTA02, BTA04, BTA07 and BTA13. These data enabled us to estimate the heritability of the phenotype using the kinship matrix between individuals computed from genotyping data. Thus, depending on the estimators considered and the method used, the heritability of anaemia control ranged from 0.09 to 0.22. Finally, an analysis of association identified an allele of the MNB42 marker on BTA04 as being strongly associated with anaemia control, and a candidate gene, INHBA, as being close to that marker.

Farmers' perceptions on trypanosomosis and trypanotolerance character of the taurine Sheko.

In the humid and subhumid tropics, trypanosomosis is an economically important zoonotic protozoan disease of the commonly kept farm animal species and their wild relatives. For example, more than 20% of the humid western and southwestern Ethiopia, which is home to more than 14 million heads of cattle, is under varying levels of trypanosomosis risk. Our study was, therefore, initiated to document farmers' perception on trypanosomosis and Sheko's trypanotolerance character. Our findings showed that trypanosomosis was the most frequently reported cattle disease in the Bench Maji Zone. Accordingly, 76.7% of the farmers reported the epidemiological importance of trypanosomosis, and they also noted that trypanosomosis on average accounted for 63.0% of annualized cattle death. The reported signs of trypanosomosis and trypanotolerance indicators were consistent with literature reports. Moreover, 66.7% of the farmers reported Sheko's trypanotolerance character. In the course of time, smallholder farmers have developed ethnoveterinary practices that are mainly used to prevent the landing of vector flies on the animal. Wet and warm seasons of the year, i.e., spring and, to some extent, the beginning of summer and autumn, were reported as peak periods of trypanosomosis risk. Therefore, this showed the need for incorporating farmers' knowledge in trypanosomosis control programs.

Targeting cattle-borne zoonoses and cattle pathogens using a novel trypanosomatid-based delivery system.

Trypanosomatid parasites are notorious for the human diseases they cause throughout Africa and South America. However, non-pathogenic trypanosomatids are also found worldwide, infecting a wide range of hosts. One example is Trypanosoma (Megatrypanum) theileri, a ubiquitous protozoan commensal of bovids, which is distributed globally. Exploiting knowledge of pathogenic trypanosomatids, we have developed Trypanosoma theileri as a novel vehicle to deliver vaccine antigens and other proteins to cattle. Conditions for the growth and transfection of T. theileri have been optimised and expressed heterologous proteins targeted for secretion or specific localisation at the cell interior or surface using trafficking signals from Trypanosoma brucei. In cattle, the engineered vehicle could establish in the context of a pre-existing natural T. theileri population, was maintained long-term and generated specific immune responses to an expressed Babesia antigen at protective levels. Building on several decades of basic research into trypanosomatid pathogens, Trypanosoma theileri offers significant potential to target multiple infections, including major cattle-borne zoonoses such as Escherichia coli, Salmonella spp., Brucella abortus and Mycobacterium spp. It also has the potential to deliver therapeutics to cattle, including the lytic factor that protects humans from cattle trypanosomiasis. This could alleviate poverty by protecting indigenous African cattle from African trypanosomiasis.

Serological characterizations of tandem repeat proteins for detection of African trypanosome infection in cattle.

Serological diagnosis is a useful method to detect African trypanosome infection in livestock animals. Currently available serological tests utilize whole parasites or crude antigens, and recombinant antigens may improve reproducibility/standardization and reduce production costs. With a goal of identifying such recombinant proteins, we computationally identified proteins with tandem repeat (TR) domain from the parasite proteomes and evaluated their potential for serological diagnosis of African trypanosome infections in cattle. Among those tested, Tbg4 demonstrated the best performance with 92% sensitivity, followed by TbbGM6 (85%), TcoGM6 (85%), Tbg2 (65%) and Tbg5 (65%). Although further evaluations such as investigating cross-reactivity to other infections are needed, our data indicate the potential of these antigens for detection of African trypanosome infection in cattle.

An evaluation of melarsomine hydrochloride efficacy for parasitological cure in experimental infection of dairy cattle with Trypanosoma evansi in Thailand.

Melarsomine hydrochloride can cure Trypanosoma evansi infection in camels at a dose of 0·25 mg/kg, but at that dose relapses occur in cattle. In our study, the efficacy of an intramuscular injection of melarsomine hydrochloride at 0·5 mg/kg was assessed in 3 normal and 3 splenectomized dairy cattle experimentally infected with a stock of T. evansi from Thailand. The animals were monitored for 5 months by haematocrit centrifugation, blood- or cerebrospinal fluid-mouse inoculation, polymerase chain reaction, the card agglutination test (CATT) for T. evansi, and the enzyme-linked immunosorbent assay­T. evansi. Parasitological and DNA tests became and remained negative just after treatment. By the end of the experiment, CATT was negative and ELISA scores were below or very close to the cut-off value. One of the splenectomized cattle died from anaplasmosis during the experiment, but tested negative for surra. It was concluded that the parasites had been cleared from the cattle, and melarsomine hydrochloride at 0·5 mg/kg can be recommended for treatment against T. evansi infection in dairy cattle in Thailand. Further work is necessary to validate the efficacy of the treatment in the event of confirmed CSF-infection.

Trypanosomosis and phenotypic features of four indigenous cattle breeds in an Ethiopian field study.

We conducted a two-part study in the native home areas of four cattle breeds, Abigar, Gurage, Horro and Sheko, in south-western Ethiopia. The first part of the study investigated livestock keeper knowledge about trypanosomosis and trypanotolerance. For each breed 60 livestock keepers were interviewed, resulting in a total of 240 interviews. The second part of the study focused on biological evidence for trypanotolerance. Blood samples of about 100 head of cattle per breed were collected during peak trypanosomosis challenge period and analyzed for packed cell volume (PCV) and parasitaemia. In addition individual body measurements of the sampled animals were taken and the keepers provided some information regarding their animals. Livestock keeper interviews revealed that trypanosomosis was considered a major problem in all areas (95-100%). Almost all Abigar livestock keepers knew how trypanosomosis is transmitted, whereas only 34-52% of the keepers of the other breeds had that knowledge. Most Sheko keepers (75%) knew of trypanotolerance and claimed to have trypanotolerant animals in their own herds. Among the other three breeds the knowledge of trypanotolerance was much less (8-18%). A majority of the keepers were interested in purchasing trypanotolerant animals. PCV was highest among Horro (26.2) and Sheko (25.1) cattle whereas Abigar had the lowest PCV (20.0). Sheko were least infected by trypanosomes (6%) and had the lowest number of trypanocidal treatments per year (1 treatment/animal and year). Abigar cattle were most infected (23%) followed by Gurage (20%) and Horro (17%). Gurage had by far the highest number of treatments per animal and year (24). There were large differences between the number of cattle perceived by the keepers to be infected, and the number detected from blood sampled, among Abigar, Gurage and Horro. Sheko livestock keepers were better at correctly diagnosing trypanosomosis in their animals. It is concluded that Sheko cattle have higher trypanotolerance attributes of the breeds investigated and a better use of this breed could improve cattle health and household welfare in tsetse-infested areas.

Membrane permeabilization by trypanosome lytic factor, a cytolytic human high density lipoprotein.

Trypanosome lytic factor (TLF) is a subclass of human high density lipoprotein (HDL) that mediates an innate immune killing of certain mammalian trypanosomes, most notably Trypanosoma brucei brucei, the causative agent of a wasting disease in cattle. Mechanistically, killing is initiated in the lysosome of the target trypanosome where the acidic pH facilitates a membrane-disrupting activity by TLF. Here we utilize a model liposome system to characterize the membrane binding and permeabilizing activity of TLF and its protein constituents, haptoglobin-related protein (Hpr), apolipoprotein L-1 (apoL-1), and apolipoprotein A-1 (apoA-1). We show that TLF efficiently binds and permeabilizes unilamellar liposomes at lysosomal pH, whereas non-lytic human HDL exhibits inefficient permeabilizing activity. Purified, delipidated Hpr and apoL-1 both efficiently permeabilize lipid bilayers at low pH. Trypanosome lytic factor, apoL-1, and apoA-1 exhibit specificity for anionic membranes, whereas Hpr permeabilizes both anionic and zwitterionic membranes. Analysis of the relative particle sizes of susceptible liposomes reveals distinctly different membrane-active behavior for native TLF and the delipidated protein components. We propose that lysosomal membrane damage in TLF-susceptible trypanosomes is initiated by the stable association of the TLF particle with the lysosomal membrane and that this is a property unique to this subclass of human HDL.

Economic assessment of the performance of trypanotolerant cattle breeds in a pastoral production system in Kenya.

Cattle are the major source of food security and income for pastoral farmers in sub-Saharan Africa. However, infectious and parasitic diseases remain a major constraint to improved cattle productivity in the region. The use of animal health economics to support decision-making on cost-effective disease control options is increasingly becoming important in the developing world. Trypanotolerant indigenous Orma/zebu cattle in a trypanosomosis-endemic area of Kenya were evaluated for economic performance using gross-margin analysis and partial-farm budgeting. Orma/zebu and Sahiwal/zebu cross-bred cattle were exposed to similar husbandry practices and monitored for growth rate, incidence of common infections (trypanosomosis, anaplasmosis, babesiosis, East Coast Fever and helminthosis) and the cost of treatment assessed. Interview questionnaires were also used to assess the preference rating of the 2 breeds. Results indicated that incidence of infection was trypanosomosis 3%, anaplasmosis 58%, babesiosis 11%, East Coast Fever 22% and helminthosis 28%, with no significant difference between breeds. The Orma/zebu and Sahiwal/zebu breeds had comparable economic benefits, hence a pastoralist in Magadi division is likely to get similar returns from both breeds. This study therefore recommends adoption of not only the Sahiwal/zebu but also the Orma/zebu breed for cattle improvement in trypanosomosis endemic areas and conservation of indigenous genetic resources.

Anti-VSG antibodies induce an increase in Trypanosoma evansi intracellular Ca2+ concentration.

Trypanosoma evansi and Trypanosoma vivax have shown a very high immunological cross-reactivity. Anti-T. vivax antibodies were used to monitor changes in the T. evansi intracellular Ca2+ concentration ([Ca2+]i) by fluorometric ratio imaging from single parasites. A short-time exposure of T. evansi parasites to sera from T. vivax-infected bovines induced an increase in [Ca2+]i, which generated their complete lysis. The parasite [Ca2+]i boost was reduced but not eliminated in the absence of extracellular Ca2+ or following serum decomplementation. Decomplemented anti-T. evansi VSG antibodies also produced an increase in the parasite [Ca2+]i, in the presence of extracellular Ca2+. Furthermore, this Ca2+ signal was reduced following blockage with Ni2+ or in the absence of extracellular Ca2+, suggesting that this response was a combination of an influx of Ca2+ throughout membrane channels and a release of this ion from intracellular stores. The observed Ca2+ signal was specific since (i) it was completely eliminated following pre-incubation of the anti-VSG antibodies with the purified soluble VSG, and (ii) affinity-purified anti-VSG antibodies also generated an increase in [Ca2+]i by measurements on single cells or parasite populations. We also showed that an increase of the T. evansi [Ca2+]i by the calcium A-23187 ionophore led to VSG release from the parasite surface. In addition, in vivo immunofluorescence labelling revealed that anti-VSG antibodies induced the formation of raft patches of VSG on the parasite surface. This is the first study to identify a ligand that is coupled to calcium flux in salivarian trypanosomes.

Anti-Trypanosoma brucei activity in Cape buffalo serum during the cryptic phase of parasitemia is mediated by antibodies.

Cape buffalo are reservoir hosts of African trypanosomes. They rapidly suppress population growth of the highly antigenically variable extracellular haemoprotozoa and subsequently maintain a cryptic infection. Here we use in vitro cultures of trypanosomes cloned from Cape buffalo blood during cryptic infection, as well as related and unrelated trypanosomes, to identify anti-trypanosome components present in cryptic-phase infection serum. Trypanosome clone-specific complement-dependent trypanolytic IgM and IgG arose after appearance of target trypanosomes during cryptic infection. Serum collected late in the cryptic phase of infection contained complement-independent growth-inhibitory IgG which varied in activity among target trypanosomes. Removal of protein A/G-binding IgG from the serum restored its capacity to support trypanosome growth in vitro. Recovered growth-inhibitory IgG reacted with the variable surface glycoprotein (VSG) of parasites most affected by it, and reacted with trypanosome common antigens, notably the endosome-restricted tomato lectin-binding glycoproteins (TL-antigens). The inclusion of purified TL-antigens in culture medium did not affect the trypanosome growth-inhibitory activity of immune Cape buffalo serum. In addition, hyperimmune rabbit IgG against TL-antigens showed little or no binding to intact trypanosomes and did not affect trypanosome growth in vitro although it did react strongly with TL-antigens and trypanosome endosomes. We conclude that antibodies, particularly clone-specific (putatively VSG-specific) antibodies are responsible for the anti-trypanosome activity of cryptic phase infection serum consistent with a dominant role in parasite control in Cape buffalo.

Cytokine mRNA profiling of peripheral blood mononuclear cells from trypanotolerant and trypanosusceptible cattle infected with Trypanosoma congolense.

To examine differences in cytokine profiles that may confer tolerance/susceptibility to bovine African trypanosomiasis, N'Dama (trypanotolerant, n = 8) and Boran (trypanosusceptible, n = 8) cattle were experimentally challenged with Trypanosoma congolense. Blood samples were collected over a 34-day period, and RNA was extracted from peripheral blood mononuclear cells. The expression levels of a panel of 14 cytokines were profiled over the time course of infection and between breeds. Messenger RNA (mRNA) transcript levels for the IL2, IL8, and IL1RN genes were significantly downregulated across the time course of infection in both breeds. There was an early increase in transcripts for genes encoding proinflammatory mediators (IFNG, IL1A, TNF, and IL12) in N'Dama by 14 days postinfection (dpi) compared with preinfection levels that was not detected in the susceptible Boran breed. By the time of peak parasitemia, a type 2 helper T cells (T(H)2)-like cytokine environment was prevalent that was particularly evident in the Boran. Increases in transcripts for the IL6 (29 and 34 dpi) and IL10 (21, 25, and 29 dpi) genes were detected that were higher in the Boran compared with N'Dama. These findings highlight the implications for using murine models to study the bovine immune response to trypanosomiasis, where in some cases cytokine expression patterns differ. Overall, these data suggest that the trypanotolerant N'Dama are more capable of responding very early in infection with proinflammatory and T(H)1 type cytokines than the trypanosusceptible Boran and may explain why N'Dama control parasitemia more efficiently than Boran during the early stages of infection.

Probabilistic order in antigenic variation of Trypanosoma brucei.

Antigenic variation in African trypanosomes displays a degree of order that is usually described as 'semi-predictable' but which has not been analysed in statistical detail. It has been proposed that, during switching, the variable antigen type (VAT) being inactivated can influence which VAT is subsequently activated. Antigenic variation proceeds by the differential activation of members of the large archive of distinct variable surface glycoprotein (VSG) genes. The most popular model for ordered expression of VATs invokes differential activation probabilities for individual VSG genes, dictated in part by which of the four types of genetic locus they occupy. We have shown, in pilot experiments in cattle, correlation between the timing of appearance of VSG-specific mRNA and of lytic antibodies corresponding to seven VSGs encoded by single-copy genes. We have then determined the times of appearance of VAT-specific antibodies, as a measure of appearance of the VATs, in a statistically significant number of mouse infections (n=22). There is a surprisingly high degree of order in temporal appearance of the VATs, indicating that antigenic variation proceeds through order in the probability of activation of each VAT. In addition, for the few examples of each available, the locus type inhabited by the silent 'donor' VSG plays a significant role in determination of order. We have analysed in detail previously published data on VATs appearing in first relapse peaks, and find that the variant being switched off does not influence which one is being switched on. This differs from what has been reported for Plasmodium falciparum var antigenic variation. All these features of trypanosome antigenic variation can be explained by a one-step model in which, following an initial deactivation event, the switch process and the imposition of order early in infection arise from the inherent activation probabilities of the specific VSG being switched on.