anti-Trypanosoma Antibodies Research Articles

Detection of anti-Trypanosoma spp. antibodies in cattle from southern Brazil.

Bovine trypanosomosis, caused by Trypanosoma vivax, is a disease that originated in Africa and currently affects cattle in several South American countries, including almost all Brazilian states. Despite the reports on T. vivax infection in southern Brazil, data on its circulation status is currently unavailable. In this study, we aimed to detect anti-Trypanosoma spp. IgG antibodies in cattle from Rio Grande do Sul and suggest areas with T. vivax transmission risk. A total of 691 serum samples from cattle in the intermediate regions of Rio Grande do Sul were analyzed using indirect immunofluorescence assay (IFA). The overall seroprevalence of anti-Trypanosoma antibodies in cattle was 24.6% (170/691). The detection rate ranged from 0-37.3%, with a high prevalence in the intermediate regions of Ijuí (37.3%), Uruguaiana (30.7%), and Passo Fundo (28.9%). Thus, these regions were suggested as possible bovine trypanosomosis risk areas due to the high seroprevalence. This is the first serological study to determine Trypanosoma spp. infection status in cattle from Rio Grande do Sul, providing data on the epidemiology of trypanosomosis in the state.

A Flow Cytometry Study of the Binding and Stimulation Potential of Inactivated Trypanosoma evansi toward Dromedary Camel Leukocytes.

Surra, a wasting disease caused by Trypanosoma evansi, is one of the major animal health burdens in camel-rearing countries, imposing significant economic losses due to reduced fertility and high mortality rates. The present study used inactivated T. evansi (from the Card Agglutination Test for Trypanosomes/Trypanosoma evansi; CATT/T. evansi) and flow cytometry to investigate their binding and activation potential toward camel leukocyte subsets. Labeling T. evansi with propidium iodide (PI) enabled their flow cytometric enumeration and identification with forward scatter (FSC; indicative for cell size) and side scatter (SSC; indicative for cell internal complexity) characteristics that are comparable with values reported for Trypanosoma cruzi. The incubation of PI-labeled non-opsonized T. evansi with camel leukocyte populations revealed that camel monocytes have the highest potential to bind T. evansi, followed by granulocytes and lymphocytes. The identification of pattern recognition receptors (PRRs) on camel immune cells and the pathogen-associated molecular patterns (PAMPs) in T. evansi that are responsible for this different binding capacity requires further studies. Stimulation of camel neutrophils with Trypanosoma evansi induced shape change, reactive oxygen species (ROS) production, and neutrophil extracellular traps (NET)-formation. To ensure that T. evansi, in the parasite concentration used in this study, is not apoptotic or necrotic to camel leukocytes, we evaluated cell apoptosis and necrosis after stimulation with T. evansi. The results revealed no impact of T. evansi stimulation for 2 h on the cell viability of camel leukocytes. Subsequent work may focus on the diagnostic employment of labeled T. evansi and flow cytometry for the detection of anti-Trypanosoma antibodies in camel serum. In addition, more efforts should be deployed to investigate the host-pathogen interaction mechanisms and the escape mechanisms of T. evansi in camels. To complete these data, further studies using the living or freshly killed parasites could also be implemented in camels and/or horses.

Seroprevalence of Trypanosoma evansi in canines by indirect fluorescent antibody test (IFAT)

An indirect fluorescent antibody technique (IFAT) was standardized to study the seroprevalence of Trypanosoma evansi among canines in and around Hyderabad, Telangana, India. The IFAT revealed an overall prevalence of anti-trypanosomal antibodies in 7.28 % (22/302) of dogs. While sex-wise incidence was not significant, the prevalence was significantly high (P=0.000) in adult dogs (10.21%) of 1–8 years of age, than in older dogs (4.21 %) of 8 years and above. No infection was recorded in puppies below one-year of age. Mongrels recorded significantly high (P=0.000) prevalence followed by crossbreds, St. Bernards, Labradors, Spitz, German Shepherds and Pomeranians in descending order. IFAT can be a simple test for screening dog populations for antibodies against T. evansi.

Development of membrane-based flow-through assay for detection of trypanosomosis in equines.

A rapid flow-through immunoassay using protein A labeled gold nanoparticles (GNPs) for the qualitative detection of trypanosomosis in equine serum samples was developed. The flow-through device consisted of antigen-coated nitrocellulose membrane fixed on absorbent pads. The GNPs based detection probe was used as the colored marker. The assay can be employed for the detection of Trypanosoma specific antibodies at 14days post-infection in the infected pooled serum samples of equines. The assay can be conducted in < 5min. The results of the flow-through assay were comparable with the methods like ELISA, dot blot assay and lateral flow assay. The developed assay showed high efficiency for detection of anti-Trypanosoma antibodies in equine sera and can be used for quick screening of serum samples under field conditions.

Parasitological, Hematological, and Immunological Response of Experimentally Infected Sheep with Venezuelan Isolates of Trypanosoma evansi, Trypanosoma equiperdum, and Trypanosoma vivax.

There are three trypanosoma species of veterinary importance in South America: (1) Trypanosoma evansi, the causative agent of derrengadera mechanically transmitted by bloodsucking insects such as tabanids, (2) Trypanosoma vivax, also mechanically transmitted by some dipteras hematophages as tabanids and/or Stomoxys, and (3) T. equiperdum, a tissue parasite adapted to sexual transmission and the causative agent of dourine, a distinctive disease that affects only Equidae. In order to evaluate the parasitological, hematological, and serological response of sheep infected with T. vivax, T. evansi, and T. equiperdum, four female sheep were experimentally infected with Venezuelan trypanosome field isolates: two T. evansi of differing virulences, one T. equiperdum; one T. vivax. Parasitemia and clinical parameters such as hematocrit, red blood cell count, hemoglobin, and body temperature were measured. T. evansi caused a chronic disease with undulant parasitemia alternating with some cryptic periods of at least 54 days, with no clinical signs. T. equiperdum, never described as infectious to ruminants, also caused a chronic disease with low undulant parasitemia. T. vivax caused an acute infection with severe anemia showing a drop of more than 70% of the hematocrit value, high fever, and rapid deterioration of physical condition, for 36 days of infection. Indirect ELISAs using crude extracts of the three species of trypanosomes as antigens were performed for detection of anti-trypanosome antibodies in sheep sera. Cross-reaction was observed between the three parasite species. These results show that sheep are susceptible to the three-trypanosome species and suggest they can act as a reservoir when sheep are raised and managed with other important livestock such as cattle, horses, buffalos, or goats. These findings are especially interesting for T. equiperdum, a species that has not been reported as infective to sheep.

Development of Lateral Flow Assay for Point-of-Care Diagnosis of Trypanosomosis in Equines

Trypanosomosis, commonly known as “Surra” in Indian subcontinent caused by Trypanosoma evansi, is a worldwide parasitic disease of wild and domestic animals. The disease is transmitted mechanically by biting and interrupted feeding of infected animals by hematophagous flies. The diagnostic methods available for this disease require high-end instruments and skilled manpower and are time-consuming. In the present investigation, we attempted an immunoassay based on gold nanoparticles for diagnosis of T. evansi infection in equines. Antibodies to horse IgG were raised in rabbits and conjugated to gold nanoparticles after purification with the protein-A column. The soluble whole cell lysate antigen was prepared from purified T. evansi parasites and coated as test line on nitrocellulose membrane laminates, along with control line to detect anti-Trypanosoma antibodies. The experimental and field serum samples were tested and evaluated to determine the sensitivity and specificity of the assay. The antigen-specific antibodies were detected in experimental serum samples between 10 and 21 days post infection by the lateral flow assay (LFA). The LFA results showed 96.3% sensitivity and 93.9% specificity comparable with enzyme-linked immunosorbent assay. The study suggests that the LFA can be used as a point-of-care diagnostic method in the field, which does not require any specialized instrument for the detection of T. evansi infection in animals.

Field investigation of Trypanosoma evansi and comparative analysisof diagnostic tests in horses from Bahawalpur, Pakistan

In order to assess the prevalence of Trypanosoma evansi, a parasitological, molecular, and serological-based investigation was carried out in randomly sampled horses (n = 375) belonging to different age groups, sexes, and localities from the district of Bahawalpur, Pakistan. The diagnostic performance of applied tests was also compared. The prevalence was recorded as 0.5% with Woo's test, 1.3% with both ITS CF/BR PCR and RoTat 1.2 PCR, and 14.4% with CATT/T.evansi. Based on CATT/T.evansi, significant differences (P ≤ 0.05) were observed for prevalence estimates according to different localities, sexes, body condition scores, and origins. A nonsignificant difference (P ≥ 0.05) was observed among different age groups and variable parity numbers. Our study declares district Bahawalpur to be a high risk area for surra and proposes the potential use of CATT/T.evansi as a field test of choice for surveys in horses. However, the status of seropositive animals should be confirmed using a more sensitive molecular tool such as the satellite DNA target. A widespread status of anti-Trypanosoma antibodies calls for control measures and further investigation of various species (camels, cattle, sheep, goats, buffaloes) inhabiting the same area to identify reservoir status.

Comparative evaluation of recombinant HSP70 (N & C-terminal) fragments in the detection of equine trypanosomosis

Trypanosomosis (Surra) is an economically important disease caused by Trypanosoma evansi which is an extracellular parasite present in the plasma, tissues and other body fluids of a wide range of hosts including domesticated animals. Currently, serological reports are based on detection of antibodies by ELISA using whole cell lysate (WCL) antigen, which has a limitation of persistence of anti-trypanosomal antibodies after successful treatment of the disease. Moreover, it has some ethical issues also like requirement of mice for in vivo maintenance of parasite for preparing the antigen. Therefore, in the present study, an attempt was made to evaluate the in vitro production of recombinant heat shock protein 70 (HSP70) for detection of antibodies in experimentally infected ponies. The amino acid sequence analysis of HSP70 revealed that N-terminal region of the protein was highly conserved while the C-terminal region was most divergent. The four different regions of HSP70 protein viz. HSP-1, HSP-2, HSP-3 and HSP-4 were cloned and expressed, among which HSP-1 (N-terminal region) & HSP-2 (C-terminal region) were truncated while HSP-3 & HSP-4 were complete C-terminal proteins. The recombinant fragments were probed with sequentially pooled experimental serum samples where antibodies were detected in these fragments from 10th day post infection till the termination of the experiment. Further, these recombinant fragments were also comparatively evaluated with WCL antigen in ELISA using experimental as well as field serum samples. It was observed that after successful treatment of infected ponies, there was a sharp fall in antibodies (within 90 days) when tested with recombinant HSP’s fragments, while antibodies persisted even after 469 days when tested against WCL antigen. The sensitivity and specificity of all HSP70 fragments were also estimated from field serum samples with reference to WCL antigen ELISA. The HSP-1 showed minimum sensitivity (41.03%) among all the recombinant fragments. Among the C-terminal fragments, maximum sensitivity was observed with the HSP-2 (61.54%) while minimum was observed with HSP-4 (48.72%). The specificity increases for recombinant fragments from N-terminal to C-terminal region of protein and maximum specificity was observed with HSP-4 fragment (91.3%).

PARASITOLOGICAL AND MOLECULAR STUDIES ON TRYPANOSOMA EVANSI OF CAMELS IN EGYPT

This study was conducted on a total of 187 one humped camels (Camelus dromedarius). Blood samples were collected from 40 camels suspected forTrypanosoma infection from farms at Giza Pyramids area. In addition, 147 samples were collected from apparently healthy camels (97 and 50 from El-Bassatin and El-Warraq abattoirs respectively) for screening of Trypanosoma species infection. All samples were examined parasitologically by Giemsa stained blood smear and haematocrit centrifugation technique, serologically by card agglutination test (CATT) for detection of anti-trypanosomal antibodies and polymerase chain reaction (PCR) for DNA amplification with aspecific primers for detection of trypanozoan parasites. Out of 187 camels, 14 camels were positive by parasitological methods with a percentage of 7.49%, 21 positive samples were detected by anti- Trypanosoma antibodies using CATT with a percentage of 11.23%. Fourteen out of the positive blood samples by parasitological and serological techniques were used for PCR amplification. Thirteen out of 14 positive blood samples were PCR-positive and one was negative by using specific primers for T. evansi minicircle EVA1 and EVA2. The results of examined camels after using the polymerase chain reaction (PCR) for detection of DNA of trypanosomes in infected camels, showed 138 bp PCR product for the specific detection of Trypanosoma evansi. It was concluded that the use of PCR, beside parasitological and serological methods, is recommended for exact diagnosis in survey and control programmes ofTrypanosoma evansi.

Serodiagnosis of bovine trypanosomosis caused by non-tsetse transmitted Trypanosoma (Duttonella) vivax parasites using the soluble form of a Trypanozoon variant surface glycoprotein antigen

Previous studies have shown that a 64-kDa antigen (p64) that was purified from the Venezuelan TeAp-N/D1 isolate of Trypanosoma (Trypanozoon) equiperdum corresponds to the soluble form of its predominant variant surface glycoprotein (VSG), and exhibited cross-reactivity with Trypanosoma (Duttonella) vivax. The course of experimental acute infections of bovines with T. vivax were followed by measuring whole anti-p64 antibodies and specific anti-p64 IgG and IgM antibodies in animal sera by indirect enzyme-linked immunosorbent assay (ELISA). The value of p64 to diagnose bovine trypanosomosis was also examined using 350 sera from healthy and T. vivax-infected cows living in a trypanosomosis-endemic and enzootic stable area, and 48 sera obtained during a trypanosomosis outbreak. Serological assays showed that ∼70–80% of the infected sera contained anti-p64 antibodies, based on the comparative immunodetection of the T. equiperdum clarified antigenic fraction used as a reference test. In the absence of a gold standard, Bayesian analysis for multiple testing estimated a sensitivity and specificity of 71.6% and 98.8%, respectively, for the indirect ELISA using p64 as antigen. An apparent prevalence of 37.7% for bovine trypanosomosis infection was also estimated with a Bayesian approach when the p64 ELISA test was used. Employing blood from acute infected cows, the indirect ELISA response against p64 was contrasted with the microhematocrit centrifuge method and analyses by polymerase chain reaction (PCR) using specific primers targeting the inter-specific length variation of the internal transcribed spacer 1 region of the 18S ribosomal gene. The efficiency of p64 for the detection of anti-trypanosome antibodies in acute infected bovines was also corroborated serologically by comparing its response to that of the Indonesian Trypanosoma evansi Rode Trypanozoon antigen type (RoTat) 1.2 VSG, which possesses high specificity and sensitivity. As expected, PCR was the best method to detect parasites and diagnose bovine trypanosomosis; however, a substantial level of concordance (Cohen’s κ=0.667) was obtained when serological tests using p64 and RoTat 1.2 VSG were compared. Additionally, an agglutination assay was designed using p64 covalently coupled to carboxylate-modified latex microparticles, which was proven here to be suitable for a fast qualitative diagnosis of bovine trypanosomosis.

The B cell adaptor molecule Bam32 is critically important for optimal antibody response and resistance to Trypanosoma congolense infection in mice.

BackgroundBam32, 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 FindingsWe 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/SignificanceCollectively, 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.

Antibody‐mediated control of Trypanosoma vivax infection fails in the absence of tumour necrosis factor

Trypanosoma vivax causes a wasting disease affecting livestock breeding and agriculture in developing countries of sub-Sahara Africa and South America. Being an extracellular parasite, control of T.vivax has been proposed to be mediated by host antibodies. However, the use of a comparative infection model of wild-type (WT) and tumour necrosis factor (TNF) knockout (TNF(-/-) ) mice shows that the latter is unable to control first-peak parasitaemia, despite the presence of specific antitrypanosome antibodies. In contrast, WT mice parasitaemia peak control coincides with a combined early onset of TNF production and induction of specific antibodies. TNF is mainly produced by liver-associated monocytes and neutrophils. In this study, no other correlation between cellular immunomodulations and peak parasitaemia control was observed, underscoring the importance of the role of TNF in the control of T.vivax infections.

Bovine trypanosomosis in the Upper West Region of Ghana: Entomological, parasitological and serological cross-sectional surveys

Baseline surveys were conducted in the Upper West Region of Ghana to assess the distribution and densities of tsetse species, as well as the prevalence of bovine trypanosomosis. The entomological survey was designed to cover the suitable tsetse habitats along the three main rivers in the study area (i.e. Black Volta, Kulpawn and Sissili). Results indicated the presence of Glossina tachinoides in all three river basins, whilst Glossina palpalis gambiensis was only found close to the southern limit of the study area. A random sampling of 1800 cattle of the West African Short Horn, Sanga and Zebu breeds from 36 randomly selected grid cells covering the study area showed substantial differences between parasitological and serological prevalences. The average parasitological prevalence was estimated at 2.5% (95% CI: 1.06–5.77) with the majority of the infections due to Trypanosoma vivax. Most of the infected cattle were found close to the major river systems. The serological prevalence, measured using Enzyme Linked Immunosorbent Assay (ELISA), test was 19% (95% CI: 14.03–25.35). Cattle with anti-trypanosomal antibodies were also found throughout the study area.

Trypanosoma brucei: Immunisation with plasmid DNA encoding invariant surface glycoprotein gene is able to induce partial protection in experimental African trypanosomiasis

Trypanosoma brucei is the etiological agent responsible for African trypanosomiasis, an infectious pathology which represents a serious problem of public health and economic losses in Sub-Saharan Africa. As one of the foremost neglected illnesses, few resources have been available for the development of vaccines or new drugs, in spite of the current therapeutical drugs showing little efficiency and high toxicity. Hence, it is obviously important to widen effective therapeutics and preventive strategies against African trypanosomiasis. In this work, we use the DNA vaccine model to evaluate immunisation effectiveness in mice challenged with Trypanosoma brucei brucei. We demonstrate that Balb/C mice immunised intramuscularly with a single dose of a DNA plasmid encoding a bloodstream-stage specific invariant surface glycoprotein (ISG) are partially protected from a lethal dose of T. b. brucei. Interestingly, the surviving animals show high levels of IgG2a anti-trypanosoma antibodies, suggesting that the Th1 response profile seems important for the induced mechanisms of immune protection.

Detection of Trypanosoma evansi infection in clinically ill cattle, buffaloes and horses using various diagnostic tests

This study was undertaken to detect Trypanosoma evansi infection in clinically ill cattle, buffaloes and horses that were suspected of 'surra' infection. Various diagnostic tests were utilized in order to assess the prevalence of infection in the clinically ill and 'suspected' animals. Animals in the Eastern Region of India were systematically studied for T. evansi infection, which had not been tested for previously. Examination of Giemsa-stained blood smears detected 5.3%, 9.4% and 40.6% infections; the mouse inoculation test detected 18.4%, 15.6% and 46.9% infections whereas an in-house ELISA detected anti-trypanosomal antibodies in 42.1%, 43.8% and 65.6% infections in clinically ill buffaloes, cattle and horses, respectively. It can be concluded that T. evansi is prevalent in cattle, buffaloes and horses in the Eastern Region of India.

Flow cytometric enumeration of parasitaemia and haematologic changes in trypanosoma-infected mice

African trypanosomiasis is a severe parasitic disease affecting both man and livestock. It is crucial to expand our fundamental knowledge of the intimate interactions between trypanosomes and their vertebrate hosts in order to develop new and efficient control strategies. The mouse model of trypanosomiasis is the most popular for research purposes because of all the logistic advantages of using this species. Studies of any aspect of trypanosomiases in the mouse systematically require the quantification of some phenotypic traits which translate its degree of resistance/susceptibility to the disease, as blood cell counts. The present study presents a methodological approach combining everyday microsampling of tail blood and its analysis by flow cytometry. The technical options and conditions permitting a fast, reliable and reproducible daily quantification of erythrocyte, reticulocyte, leucocyte and trypanosome counts in the inoculated mouse were established. The protocol proposed allows the multiplication of blood samplings without being exposed to the time-consuming constraint of visual countings, without causing iatrogenic blood cell alterations in the mouse and without requiring specific anti-trypanosome antibodies.

Effects of Exogenous Transforming Growth Factor β onTrypanosoma congolenseInfection in Mice

The socioeconomic implications of trypanosomosis in sub-Saharan Africa and the limitations of its current control regimes have stimulated research into alternative control methods. Considering the pro- and anti-inflammatory properties of transforming growth factor beta1 (TGF-beta1) and its potential to enhance immunity against protozoan parasites, we examined the effects of intraperitoneally delivered TGF-beta1 in C57BL/6 mice infected with Trypanosoma congolense, the hemoprotozoan parasite causing nagana in cattle. A triple dose of 10 ng TGF-beta1 significantly reduced the first parasitemic peak and delayed mortality of infected mice. Furthermore, exogenous TGF-beta1 significantly decreased the development of trypanosome-induced anemia and splenomegaly. The apparent TGF-beta1-induced antitrypanosome protection, occurring mainly during the early stage of infection, correlated with an enhanced parasite antigen-specific Th1 cell response characterized by a skewed type I cytokine response and a concomitant stronger antitrypanosome immunoglobulin G2a antibody response. Infected TGF-beta1-pretreated mice exhibited a significant reduction in the trypanosome-induced hyperexpansion of B cells. Furthermore, evidence is provided herein that exogenous TGF-beta1 activates macrophages that may contribute to parasite control. Collectively, these data indicate that exogenous TGF-beta1 is immunostimulative, inducing partial protection against T. congolense infection, possibly through mechanisms involving innate immune responses.

The development of an enzyme-linked immunosorbent assay for Trypanosoma vivax antibodies and its use in epidemiological surveys

There are data indicating that the distribution of Trypanosoma vivax in the Brazilian territory is expanding with potential to reach other areas, where the vectors are present. The detection of anti-trypanosomal antibodies in serum provides important information of the trypanosomal status in cattle herds. For this reason, an enzyme-linked immunosorbent assay (Tv-ELISA-Ab) with crude antigen from one Brazilian isolate of T. vivax was developed and evaluated. The sensitivity and specificity were respectively 97.6 and 96.9%. In the evaluation of cross-reactions, three calves inoculated with T. evansi trypimastigotes blood forms showed optical densities (OD) under the cut-off during the whole experimental period, except one at 45 days post-inoculation. With relation to Babesia bovis, B. bigemina, and Anaplasma marginale, which are endemic hemoparasites in the studied area, the cross-reactions were shown to be 5.7, 5.3, and 1.1%, respectively. The first serological survey of Pantanal and state of Pará showed that T. vivax is widespread, although regions within both areas had significantly different prevalences. Therefore, this Tv-ELISA-Ab may be a more appropriate test for epidemiological studies in developing countries because the diagnostic laboratories in most countries may be able to perform an ELISA, which is not true for polymerase chain reaction.

Trypanosomiasis in Venezuelan water buffaloes: association of packed-cell volumes with seroprevalence and current trypanosome infection

The seroprevalence of trypanosomiasis and the prevalence of current trypanosome infection in water buffaloes from the most important livestock areas of Venezuela were evaluated by IFAT and the microhaematocrit centrifugation technique, respectively. The usefulness of a PCR-based assay for identifying the trypanosome species in the buffaloes was also evaluated. Of the 644 animals investigated, 40 (6.2%) were found infected with trypanosomes by blood centrifugation, and 196 (30.4%) were found positive for anti-trypanosome antibodies, by IFAT. The results of the PCR-based assay indicated that 92.5% of the animals with current infections were infected with Trypanosoma vivax and the rest with T. theileri (the first molecular confirmation of T. theileri in Venezuelan water buffaloes). The national programme to treat and prevent trypanosome infections in the buffaloes does not appear to be meeting with great success, even though it is focused on T. vivax. Although the level of parasitaemia was categorized as low for 28 (70%) of the infections detected (and packed-cell volumes appeared to be unassociated with IFAT result, and uncorrelated, in the infected animals, with level of parasitaemia), the 40 infected buffaloes had a significantly lower mean packed-cell volume than the uninfected animals (P<0.05). Farmers should therefore be made aware of the probability of trypanosome-attributable losses in buffalo productivity.

Pyroglutamyl peptidase type I from Trypanosoma brucei: a new virulence factor from African trypanosomes that de-blocks regulatory peptides in the plasma of infected hosts

Peptidases of parasitic protozoans are emerging as novel virulence factors and therapeutic targets in parasitic infections. A trypanosome-derived aminopeptidase that exclusively hydrolysed substrates with Glp (pyroglutamic acid) in P1 was purified 9248-fold from the plasma of rats infected with Trypanosoma brucei brucei. The enzyme responsible was cloned from a T. brucei brucei genomic DNA library and identified as type I PGP (pyroglutamyl peptidase), belonging to the C15 family of cysteine peptidases. We showed that PGP is expressed in all life cycle stages of T. brucei brucei and is expressed in four other blood-stream-form African trypanosomes. Trypanosome PGP was optimally active and stable at bloodstream pH, and was insensitive to host plasma cysteine peptidase inhibitors. Native purified and recombinant hyper-expressed trypanosome PGP removed the N-terminal Glp blocking groups from TRH (thyrotrophin-releasing hormone) and GnRH (gonadotropin-releasing hormone) with a k(cat)/K(m) value of 0.5 and 0.1 s(-1) x microM(-1) respectively. The half-life of TRH and GnRH was dramatically reduced in the plasma of trypanosome-infected rats, both in vitro and in vivo. Employing an activity-neutralizing anti-trypanosome PGP antibody, and pyroglutamyl diazomethyl ketone, a specific inhibitor of type I PGP, we demonstrated that trypanosome PGP is entirely responsible for the reduced plasma half-life of TRH, and partially responsible for the reduced plasma half-life of GnRH in a rodent model of African trypanosomiasis. The abnormal degradation of TRH and GnRH, and perhaps other neuropeptides N-terminally blocked with a pyroglutamyl moiety, by trypanosome PGP, may contribute to some of the endocrine lesions observed in African trypanosomiasis.