First report of molecular characterization and phylogenetic analysis of RoTat 1.2 VSG of Trypanosoma evansi from equine isolate

Rotat 1.2 variant surface glycoprotein (VSG) is considered to be an important VSG expressed in most of the isolates of Trypanosoma evansi. This makes the molecule an important candidate for both molecular- and serological-based detection of surra. There are ample reports of existence of this gene in isolates from cattle, buffalo, and camel across the world. Of late, there are reports of its absence from a fewer isolates of T. evansi of murine and wildlife origin. Search of literature revealed no reports from horses. The present communication presents the first report of molecular cloning and characterization of Rotat 1.2 VSG from horse isolate of T. evansi from semi-arid region of India. Alongside, the gene was compared with various other isolates across the world. Interestingly, the isolate was found to be closer to camel isolates from Egypt than the other known isolates from India and Kenya.

     

A biochemical and immunological comparative study on Trypanosoma equiperdum and Trypanosoma evansi

Trypanosoma equiperdum and Trypanosoma evansi were purified by three or four cycles of low-speed centrifugation and final filtration through DEAE cellulose. The purified trypanosomes were used in comparative biochemical and immunological studies. Comparative polypeptide pattern analysis revealed that T. equiperdum showed 21 polypeptide bands, whose M _r ranged from >200 to 14.8 kDa. T. evansi showed 25 polypeptide bands in the M _r range 97–14.8 kDa. The main differences were associated with the presence of secondary bands, relative intensity and the number of bands. Both species gave seven glycoprotein bands; those of 97 and 68 kDa were present in T. equiperdum but absent in T. evansi. Bands of 61 and 28 kDa were present in T. evansi but not in T. equiperdum. Anti-T. equiperdum sera recognized four homologous antigens and cross-reacted with three antigens of T. evansi. Anti-T. evansi sera recognized three homologous antigens and cross-reacted with four T. equiperdum antigens. Four identical proteolytic protease bands were present for both species, while only one surface protein was detected for each species: 66 kDa for T. equiperdum and 62 kDa for T. evansi.     

Effect of tea tree oil (Melaleuca alternifolia) on the longevity and immune response of rats infected by Trypanosoma evansi

This study aimed to evaluate the effect of tea tree oil (TTO – Melaleuca alternifolia) on hepatic and renal functions, and the immune response of rats infected by Trypanosoma evansi. A pilot study has shown that rats treated with TTO orally (1 ml kg⁻¹) had increased survival rate without curative effect. In order to verify if increased longevity was related to a better immune response against T. evansi when using tea tree oil, a second experiment was conducted. Thus, twenty-four rats were divided into four groups. The groups A and B were composed of uninfected animals, and the groups C and D had rats experimentally infected by T. evansi. Animals from the groups B and D were treated orally with TTO (1 ml kg⁻¹) for three days. Blood samples were collected to verify humoral response analysis for immunoglobulins (IgA, IgM, IgE, and IgG) and cytokines (TNF-α, INF-γ, IL-1, IL-6, IL-4, and IL-10) at days 0, 3, 5 and 15 post-infection (PI). TTO treatment caused changes in the immunoglobulins and cytokines profile, as well as the course of T. evansi infection in rats. It was found that the TTO was not toxic, i.e., hepatic and renal functions were not affected. Therefore, it is possible to conclude that TTO influences the levels of inflammatory mediators and has trypanocidal effect, increasing life expectancy of rats infected by T. evansi.     

Absence of host proteins from the surface of Trypanosoma vivax of cattle

Trypanosoma vivax (EATRO 1721) organisms were isolated by DEAE-cellulose chromatography from blood of an experimentally infected calf. Attempts to agglutinate the purified trypanosomes with a rabbit antiserum against whole bovine serum or antisera monospecific for bovine IgG1, IgG2, IgM, complement component C3 or albumin were unsuccessful. The trypanosomes, however, were agglutinated by immune sera of four different calves chronically infected with T. vivax (EATRO 1721). It was concluded that T. vivax organisms purified by DEAE-cellulose chromatography from blood of cattle do not have bovine serum proteins on their surface.     

Effect of isometamidium on infections by Trypanosoma vivax and T. Evansi in experimentally-infected animals

Assays dealing with the therapeutic and prophylactic activity of isometamidium on experimental infections by Trypanosoma vivax and T. evansi were carried out. The drug was found to be highly effective against T. vivax infection in sheep and cattle in which periods of protection ranging from 118 to 195 days were achieved. No complete effects against infection by T. evansi were observed. The drug was well tolerated in sheep and cattle while side-effects were noted in treated mares. It was concluded that isometamidium could be used to prevent damage and economical losses caused by T. vivax in Venezuela.     

Comparative Evaluation of the Antibody-detection ELISA Technique Using Microplates Precoated with Denatured Crude Antigens from Trypanosoma congolense or Trypanosoma vivax

Two FAO/IAEA indirect enzyme-linked immunosorbent assays (ELISA), which use microplates precoated with denatured crude Trypanosoma congolense or Trypanosoma vivax antigen for detecting anti-trypanosomal antibodies in bovine sera, were evaluated for their sensitivity, specificity and positive and negative predictive values, using 320 Ugandan field samples (known negative sera, n = 80; known positive sera, n = 80; cattle herds where control of tsetse and trypanosomosis was practised, n = 80; and cattle herds where there was no such control, n = 80). Cut-off points of 30% and 25% positivity were determined for the T. congolense and T. vivax assays, respectively, using a modified ROC (receiver operating characteristic) analysis. The T. congolense assay had estimated diagnostic sensitivity and specificity of 63.7% and 57.5%, respectively, while the T. vivax assay had estimated diagnostic sensitivity and specificity of 81.3% and 81.3%, respectively. The two assays conducted in parallel had estimated diagnostic sensitivity and specificity of 82.5% and 88.7%, respectively. Using the sera from the cattle in the area with control (detected prevalence of trypanosomosis 0%), both the T. congolense and T. vivax assays had negative and positive predictive values of 100% and 0%, respectively. Using the sera from the cattle in the area without control (detected prevalence of trypanosomosis 15%), the T. congolense assay had negative and positive predictive values of 91% and 33%, respectively, and the T. vivax assay had negative and positive predictive values of 93% and 27%, respectively. The T. congolense assay was in fair agreement with the buffy coat technique (BCT) ( = 0.25), while the T. vivax assay was in substantial agreement with the BCT ( = 0.625), and both assays conducted in parallel were in substantial agreement with the BCT ( = 0.708). Both assays were found to be proficient and suitable for the diagnosis of bovine trypanosomosis, especially when used in parallel.     

A Preliminary Comparative Study of a Dipstick Colloidal Dye Immunoassay and Two Antigen-detection ELISAs for Diagnosis of Trypanosoma evansi Infection in Cattle

A dipstick colloidal dye immunoassay (DIA) was developed for the field diagnosis of Trypanosoma evansi infection using affinity-purified polyclonal antibodies (PcAbs) and the monoclonal antibody (McAb) 8B9. PcAbs were adsorbed onto Palanil Red dye particles and used as dye reagents. Dipsticks were dotted with four different antibodies; normal rabbit and mouse IgGs as negative controls, and anti-T. evansi PcAb and McAb 8B9, which capture trypanosome antigens in the tested samples. Since the dye reagent bound to the captured antigens, the presence of coloured dots on the dipstick identified trypanosome infections. The sensitivity of the DIA was compared with two antigen detection ELISAs (Ag-ELISA); one was PcAb-based and the other was based on a combination of the same Mc- and PcAbs as were employed for the DIA. With a positive serum, the DIA detected trypanosomal antigen up to a dilution of 1:500 for both the PcAb and McAb dots, at which dilution the PcAb- and combination-based Ag-ELISA gave positive OD readings of 0.13 and 0.36, respectively. When 124 field sera were tested, circulating antigens were detected in 51 (41%) samples by the DIA, and 76 (61%) and 49 (40%) samples by the PcAb- and combination-based Ag-ELISAs respectively, of which 48 (63%) and 34 (69%) were also positive by the DIA.     

Expressed truncated N-terminal variable surface glycoprotein (VSG) of Trypanosoma evansi in E. coli exhibits immuno-reactivity

The variant surface glycoprotein (VSG) of trypanosome is an important part of its body surface coat, which is expressed in early, middle and late stages of infection contributing a major diagnostic value. In the present study, the 5' end of the partial VSG gene sequences (681 bp) encoding N-terminal protein of RoTat 1.2 VSG (227 amino acid) was amplified, cloned into pET32a vector, and expressed in prokaryotic system. The fused His-tagged expressed VSG protein (43 kDa) of the Trypanosoma evansi was characterized in SDS-PAGE and immunoblotting using hyperimmune/immune sera raised against buffalo, dog, lion and leopard isolates of T. evansi. The expressed protein remained immunoreactive with all the sera combinations. The animals immunized with whole cell lysate or recombinant protein showed similar antibody reactions in ELISA and CATT (Card Agglutination Test for Trypanosomiasis). This study suggests the expressed recombinant truncated VSG is having its importance for its possible use in sero-diagnosis of surra.     

Follicular degeneration in the ovaries of goats experimentally infected with Trypanosoma vivax from the Brazilian semi-arid region

Infection by Trypanosoma vivax and other African trypanosomes plays an important role in reproductive disorders in male and female livestock. Outbreaks of T. vivax in the semi-arid region of northeastern Brazil are characterized by wasting disease in cattle, sheep and goats with hematological, cardiac and nervous compromises in addition to reproductive failures. Similar to reports from Africa, we previously observed a reduction in fertility rates and severe testicular degeneration and epididymitis in male sheep infected with T. vivax from this region. Although anestrus is frequently reported in goats and sheep infected with T. vivax, the effects of this infection on the female reproductive organs need clarification. In this study, we addressed this issue through a histopathological evaluation of ovarian follicular morphology and classification in goats experimentally infected with a T. vivax isolate from the Brazilian semi-arid region. The infected animals presented typical clinical signs of trypanosomosis by T. vivax, including anemia, hyperthermia, pallor of the mucous membranes, enlarged lymph nodes, and progressive loss of weight. All the infected goats remained anestrus throughout the experimental period and exhibited important disturbances in the ovaries, evidenced by reduced size and a smooth surface without follicles or corpora lutea, and abnormal follicular development. In addition, through PCR, we detected T. vivax DNA in the ovarian tissues of the infected goats. Our findings contributed to understand the female reproductive failure associated with trypanosomosis caused by T. vivax.     

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.     

The chemotactic response of murine peritoneal exudate cells to Trypanosoma brucei

A modified Boyden technique was used to assess the chemotactic activity of unstimulated mouse peritoneal exudate cells in the presence of Trypanosoma brucei. Live parasites, the soluble fraction obtained from disrupted trypanosomes, and specific mouse hyperimmune T. brucei sera were unable to generate chemotactic activity. Complexes of whole parasites and hyperimmune sera gave similar control values, whereas significant activity was obtained using a mixture of live trypanosomes and untreated mouse plasma. Immune complexes prepared by pre-incubation of soluble trypanosome fractions with either untreated or heat inactivated (56°C for 30 min) hyperimmune sera, or with mouse plasma, also resulted in enhanced chemotactic activity.     

Relationship between splenic sequestration and thrombocytopenia in Trypanosoma evansi infection in rats

Trypanosoma evansi infections in domestic animals are characterized by anemia and thrombocytopenia. The cause of the platelets decrease is unknown, but researchers suggest that thrombocytopenia may result from damage of the bone marrow, reduced survival of platelets, auto-immune thrombocytopenia, disseminated intravascular coagulation and splenic sequestration. Some of these causes have already been tested by our research group and found to be unrelated. Therefore, this study has the objective of testing the hypothesis that splenic sequestration might be responsible for thrombocytopenia in T. evansi-infected rats. A total of 28 rats assigned to four groups were used in the experiment. Group A rats were splenectomized and infected with T. evansi, group B rats were infected with T. evansi, group C rats were splenectomized, but not infected and group D rats were normal controls. Five days post-infection all rats were anesthetized and blood was collected in order to measure the number of circulating platelets, fibrinogen levels, prothrombin time (PT) and activated partial thromboplastin time (aPTT). The spleens of groups B and D were weighed at necropsy. The infected animals (groups A and B) showed a significant reduction in platelets and increased PT and aPTT when compared to negative control groups (groups C and D). Animals from group A showed increased levels of fibrinogen. The mean weight of spleen differed between group B (2.62 g) and group D (0.55 g). It was concluded that there is no relationship between thrombocytopenia and splenic sequestration in infection by T. evansi.     

Detection of <Emphasis Type="Italic">Trypanosoma vivax</Emphasis> DNA in semen from experimentally infected goats

The present work aimed to investigate the presence of T. vivax DNA in the semen of experimentally infected goats. Twelve male goats native to the Brazilian Northeast, adults, were randomly assigned to two experimental groups: the infected group consisting of six goats infected intravenously with 0.5 mL of blood containing approximately 1.25?×?10⁵ trypomastigotes of T. vivax, and a control group composed of six uninfected goats. After the infection, clinical examinations aiming to evaluate rectal temperature, parasitemia and hematocrit were performed. Semen samples were collected from goats by electroejaculation on the 7th, 14th and 21st days post-infection (dpi). The recombinant DNA-encoding gene encoding the L-like-specific gene for T. vivax. The infection was characterized by increased rectal temperature, high parasitemia and significant reduction of hematocrit values. Results for T. vivax DNA detection using TviCatL-PCR were positive in all semen samples from the infected group collected on 7th, 14th and 21st dpi. The presence of T. vivax DNA in 7th dpi suggests the early invasion of the parasite in the reproductive organs. Also, the finding of T. vivax DNA in all periods analyzed may suggest the continued elimination of the parasite in the semen, which may increase the chances of sexual transmission. Thus, T. vivax DNA is recorded for the first time in the semen of infected goats. Thus, these data are of great importance, since the detection of the T. vivax genetic material in the semen may point to the possibility that the parasite may be transmitted through the sexual pathway.     

PCR amplification of RoTat 1.2 VSG gene in Trypanosoma evansi isolates in Kenya

A direct card agglutination test for Trypanosoma evansi, CATT/T. evansi based on the predominant variable antigen-type (pVAT) RoTat 1.2 was evaluated previously in the field in Isiolo District, Kenya. Sixteen out of 51 (31.4%) parasitologically positive camels were negative by the antibody detection test. In the present study, trypanosomes isolated from the camels were analysed in an attempt to determine the cause of the false negative results of CATT/T. evansi. A total of 20 field isolates comprised 16 stocks from camels that were negative by CATT/T. evansi, and 4 from CATT/T. evansi-positive camels. In addition, 15 known T. evansi and four T. brucei were used as reference. Purified DNA samples were tested using an established RoTat 1.2-based polymerase chain reaction (PCR) that yields a 488 bp product for the specific detection of T. evansi. Antibodies to RoTat 1.2 variant surface glycoprotein (VSG) were used in Western blotting to detect RoTat 1.2 VSG linear epitopes. Results of PCR and Western blot showed that the 16 stocks isolated from CATT/T. evansi-negative camels fell into three groups. In Group 1, both the RoTat 1.2 VSG gene and the VSG were absent in three stocks. In five trypanosome stocks in Group 2, the RoTat 1.2 VSG gene was detected, but Western blot was negative indicating absence of the expressed VSG. Five other stocks containing the RoTat 1.2 VSG gene were also in this group. The RoTat 1.2 VSG gene was detected and Western blot was positive in all four trypanosome stocks in Group 3. All four stocks from CATT/T. evansi-positive camels contained the RoTat 1.2 VSG gene and the expressed VSG. The reference T. evansi KETRI 2479 lacked the RoTat 1.2 VSG gene and there was no immune reactivity detected by Western blot. The rest of the reference T. evansi stocks examined contained the RoTat 1.2 VSG gene. All the four T. brucei samples examined were negative by PCR and Western blot. In conclusion, this study showed that the RoTat 1.2 VSG gene was absent from some T. evansi trypanosomes in Kenya.     

IgG antibodies from dourine infected horses identify a distinctive Trypanosoma equiperdum antigenic pattern of low molecular weight molecules

Diagnosis and control of dourine is strongly based on serological evidence, but knowledge of the humoral response of horses during infection is limited. In this study we developed a chemiluminescent immunoblotting (cIB) assay to characterise the Trypanosoma equiperdum antigen pattern recognised by IgGs from naturally or experimentally dourine-infected horses and analyse the kinetics of IgG humoral response following the infection. One compounding factor is that sera from uninfected animals often cross-react with T. equiperdum antigens. Development of the cIB assay was based on the hypothesis that serum IgGs from healthy and infected animals recognise different T. equiperdum antigen patterns. We used sera from 8 naturally infected horses which had recovered from Italian outbreaks and 2 experimentally infected mares. In addition, sera from 10 healthy control animals, eight of which were CFT positive but IFA negative for dourine, were collected from disease free regions. Sera were compared by the complement fixation test (CFT), indirect immune fluorescence (IFA) and the cIB assay.cIB analysis revealed that IgGs from infected horses, in contrast to IgGs from healhty horses, specifically recognise a T. equiperdum antigenic profile with low molecular weight bands ranging between 16 and 35 kDa. A time course experiment indicated that IgGs specific for the 16–35 kDa parasite protein fraction appear 17 days post-infection. The cIB assay confirmed all ten infected animals as positive and all controls as negative. This study demonstrated that analysis of IgGs by cIB can provide clear confirmation of trypanosome infection in horses, suggesting that this technique can be applied as a confirmatory serological test for dourine infection.     

Host stress physiology and Trypanosoma haemoparasite infection influence innate immunity in the woylie (Bettongia penicillata)

Understanding immune function is critical to conserving wildlife in view of infectious disease threats, particularly in threatened species vulnerable to stress, immunocompromise and infection. However, few studies examine stress, immune function and infection in wildlife. We used a flow cytometry protocol developed for human infants to assess phagocytosis, a key component of innate immunity, in a critically endangered marsupial, the woylie (Bettongia penicillata). The effects of stress physiology and Trypanosoma infection on phagocytosis were investigated. Blood and faecal samples were collected from woylies in a captive facility over three months. Trypanosoma status was determined using PCR. Faecal cortisol metabolites (FCM) were quantified by enzyme-immunoassay. Mean phagocytosis measured was >90%. An interaction between sex and FCM influenced the percentage of phagocytosing leukocytes, possibly reflecting the influence of sex hormones and glucocorticoids. An interaction between Trypanosoma status and FCM influenced phagocytosis index, suggesting that stress physiology and infection status influence innate immunity.     

伊氏锥虫在小鼠体内抗原变异规律及免疫保护试验

为了探索伊氏锥虫抗原的变异规律及其用于免疫预防的可能性，首先对伊氏锥虫安微株单虫克隆2个早期变异体ShTatl.1、ShTat1.2采用蛋白酶抑制剂TLCK处理，分离纯化这2种变异体的VSG，用ShTat1.1 VSG免疫KM小鼠，ShTat1.1锥虫攻击免疫鼠后6d分离锥虫，经间接免疫荧光试验（IFT）和班点酶标记试验（Dot-EIA)鉴定为ShTat1.2，说明同一克隆锥虫感染兔，小鼠第1次发生抗原变异后产生的变异体相同。依据这一规律，设计了免疫预防保护试验，试验小鼠分3组：一组为未免疫对照组，一组为ShTat1.1VSG单一抗原免疫组，另一组为ShTat1.1 VSG,ShTat1.2 VSG混合免疫抗原免疫组，各组均以ShTat1.1锥虫攻击，结果ShTat1.1 VSG ShTat1.2VSG免疫组小鼠全部获得保护，单用ShTat1.1 VSG免疫组小鼠和未免疫小鼠血中全部出虫、死亡，前者比后者出虫时间、存活时间延长。提示运用伊氏锥虫抗原变异这一规律设计的这种复合抗原，能激发宿主克服虫体抗原变异对免疫保护的干扰。     

Canine Trypanosoma evansi infection introduced into Germany

A 9‐year‐old male Jack Russell Terrier with a history of travel to Thailand was presented with chronic lethargy, weight loss, unilateral anterior uveitis, pancytopenia, hyperglobulinemia, and proteinuria. Numerous trypomastigotes were found on a blood smear, and using molecular methods the parasite was identified as Trypanosoma evansi. After initial response to treatment, the dog experienced a relapse with central neurologic signs 88 days after initial presentation and died. Antibodies to T evansi were detected in both serum and cerebrospinal fluid (CSF) using a card agglutination test (CATT/T evansi), and PCR analysis of CSF for T evansi was positive. Findings at necropsy included marked non‐purulent meningoencephalitis. Chronic infection with T evansi in a dog that returned to Germany following international travel highlights the risk associated with introduction of foreign animal diseases to Europe and the possibility of these infections becoming endemic. Detection of chronic infection and curative therapy of trypanosomiasis are challenging, and infection is usually fatal in the dog.     

Isolation of two antigens from Trypanosoma evansi that are partially responsible for its cross-reactivity with Trypanosoma vivax

In Venezuela, two non-tsetse transmitted trypanosomes, Trypanosoma evansi and Trypanosoma vivax, are the major etiological agents of animal trypanosomosis. Rodents can be experimentally infected with T. evansi in order to obtain enough parasites to prepare antigens for serological tests. On the contrary, the production of T. vivax antigens is a limiting factor in most laboratories. Since T. evansi and T. vivax have exhibited a very high immunological cross-reactivity, we have focused on the identification of antigens from T. evansi responsible for this phenomenon. The predominant 64 kDa glycosylated cross-reacting antigen was recently purified from the TEVA1 T. evansi Venezuelan isolate [Parasitology 124 (2002) 287]. Here, we purified two additional cross-reacting antigens with molecular masses of approximately 51 and 68 kDa from the cytosolic fraction of the same T. evansi isolate, by sequential chromatography on DEAE-sepharose and sephacryl S-300. Sera obtained from animals infected with T. evansi or T. vivax recognized both purified proteins, suggesting their potential use as diagnostic reagents.     

Application of crude and recombinant ELISAs and immunochromatographic test for serodiagnosis of animal trypanosomosis in the Umkhanyakude district of KwaZulu-Natal province,South Africa

A total of 231 serum samples were collected from sheep (n=9), goats (n=99) and cattle (n=123) in northeastern KwaZulu-Natal, South Africa. Trypanosome infection was detected using Trypanosoma brucei brucei crude antigen (TbbCA) and T. congolense crude antigen (TcoCA) ELISA assays. Recombinant antigen (T. evansi GM6 which consisted of 4 repeat domains, TeGM6-4r) ELISA and immunochromatographic test (ICT) were also used. Crude antigen ELISA, TeGM6-4r-ELISA and ICT detected 27.3%, 29% and 19.9% of trypanosome seropositive samples, respectively. Trypanosome infection prevalence in cattle and goats was 35.8–46.3% and 0–9.1%, respectively. Out of 9 sheep serum samples, 2–4 sera (22.2–44.4%) were positive. The detection performance of crude and recombinant antigen ELISAs was relatively similar (K=0.6–0.7); both are recommended for reference diagnosis and large scale epidemiological surveys. There is potential application for ICT in on-site diagnosis, but its sensitivity should be improved.