Identification of Trypanosome Proteins in Plasma from African Sleeping Sickness Patients Infected with T. b. rhodesiense

Control of human African sleeping sickness, caused by subspecies of the protozoan parasite Trypanosoma brucei, is based on preventing transmission by elimination of the tsetse vector and by active diagnostic screening and treatment of infected patients. To identify trypanosome proteins that have potential as biomarkers for detection and monitoring of African sleeping sickness, we have used a ‘deep-mining” proteomics approach to identify trypanosome proteins in human plasma. Abundant human plasma proteins were removed by immunodepletion. Depleted plasma samples were then digested to peptides with trypsin, fractionated by basic reversed phase and each fraction analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This sample processing and analysis method enabled identification of low levels of trypanosome proteins in pooled plasma from late stage sleeping sickness patients infected with Trypanosoma brucei rhodesiense. A total of 254 trypanosome proteins were confidently identified. Many of the parasite proteins identified were of unknown function, although metabolic enzymes, chaperones, proteases and ubiquitin-related/acting proteins were found. This approach to the identification of conserved, soluble trypanosome proteins in human plasma offers a possible route to improved disease diagnosis and monitoring, since these molecules are potential biomarkers for the development of a new generation of antigen-detection assays. The combined immuno-depletion/mass spectrometric approach can be applied to a variety of infectious diseases for unbiased biomarker identification

Scientific Opinion on monitoring procedures at slaughterhouses for bovines

This scientific opinion proposes toolboxes of welfare indicators for developing monitoring procedures at slaughterhouses for bovines stunned with penetrative captive bolt or slaughtered without stunning. In particular, the opinion proposes welfare indicators together with their corresponding outcomes of consciousness, unconsciousness or death. In the case of slaughter with captive bolt stunning, the opinion proposes a toolbox of indicators and the outcomes to be used to assess consciousness in bovine animals at three key stages of monitoring: (a) after stunning and during shackling and hoisting; (b) during neck cutting or sticking; and (c) during bleeding. For slaughter of bovines without stunning, a set of indicators and outcomes are proposed in another toolbox to be used for (a) assessing unconsciousness, before releasing bovines from restraint; and (b) confirming death before carcass dressing begins. Various activities—including a systematic literature review, an online survey and stakeholders’ and hearing experts’ meetings—were conducted to gather information about the specificity, sensitivity and feasibility of the indicators that can be included in the toolboxes. The frequency of checking differs according to the role of each person responsible for ensuring animal welfare. Personnel performing stunning, shackling, hoisting and/or bleeding will have to check all the animals and confirm that they are not conscious following stunning or before release from the restraint. For the animal welfare officer, who has the overall responsibility for animal welfare, a mathematical model for the sampling protocols is proposed, giving some allowance to set the sample size of animals that he/she needs to check at a given throughput rate (total number of animals slaughtered in the slaughterhouse) and tolerance level (number of potential failures). Finally, different risk factors and scenarios are proposed to define a ‘normal’ or a ‘reinforced’ monitoring protocol, according to the needs of the slaughterhouse

Molecular characterization of bovine trypanosomes from the Kachia Grazing Reserve, north-west Nigeria

Primers (Kin 1 and Kin 2) designed to amplify the internal transcribed spacer1 (ITS1) of ribosomal deoxyribonucleic acid (rDNA) and serve as a universal diagnostic test for all pathogenic trypanosomes were further evaluated for polymerise chain reaction (PCR) diagnosis and characterization of liveestock trypanosomes. Blood samples collected from 121 cattle and 3 sheep from the Kachia Grazing Reserve, north-west Nigeria in February 2005 were examined for the presence of trypanosomes using the Buffy coat technique and Giemsathin bloodfilms. 36 cattle were found positive for trypanosomes: 33 with Trypanosoma vivax, 2 with T. congolense and one mixed infection of T. congolense and T. vivax; in sheep 2 of T. vivax and one T. brucei infection. DNA extracted from all positive samples and 86 negative samples were subjected to PCR amplification using the ITS1 primers. The PCR assay allowed detection and characterization of three Trypanosoma livestock species namely T. vivax, 13 of 33 was positive; T. congolense forest, 1 of 2 and 1 T. b. brucei. The sizes of base pairs were 147 bp, 710 bp and 480 bp. respectively. The one mixed infection was detected the way it was as T. congolense and T. vivax plus another mixed infection of T. congolense and T. vivax, which was not revealed by the buffy coat. In the 86- trypanosome negative samples, 82 (95.3%) were still negative while 4 (4.7% were detected with T. vivax infection. Overall, the sensitivity of the ITS1 primers PCR detection was 42.9% and specificity 95.3%. The study concludes that the ITS1 primers are adequate for type characterization of livestock trypanosomes through a single PCR, thus saving cost. For diagnostic purposes, improvement in primer designs for enhancing T. vivax detection in field samples is suggested. Keywords: polymerase chain reaction, primers (Kin 1 and Kin 2), internal transcribed spacer 1(ITS1), ribosomal DNA, African animal trypanosomes. Nigerian Journal of Parasitology Vol. 29 (2) 2008: pp. 98-10

Trypanosoma congolense: A comparison of T-cell-mediated responses in lymph nodes of trypanotolerant and trypanosusceptible cattle during primary infection

A comparison of T-cell-mediated immune responses in trypanotolerant N'Dama and susceptible Boran cattle during primary infection with tsetse-transmittedTrypanosoma congolensewas conducted to assess whether different patterns of T-cell activation occurred during trypanosome infection. Proliferation and IFN-γ synthesis in response to trypanosome antigens and to the mitogen Con A were measured in LNC before infection and 10 and 35 days postinfection. Phenotypic analysis of LNC was also carried out. No significant differences in thein vitroproliferation of LNC to VSG, to hsp70/BiP, or to Con A were detected between the breeds. In contrast, IFN-γ production in response to Con A was higher in Boran cattle at 35 days p.i. A reduction in the number of CD2+ and CD4+ T-cells and an increase in the percentage of B-cells, CD8+ T-cells, and γδ T-cells during infection in both N'Dama and Boran was revealed by cytofluorimetric analysis of lymph node cells

Trypanosoma congolense: B-lymphocyte responses differ between trypanotolerant and trypanosusceptible cattle

Trypanosomiasis is a serious constraint to livestock production in sub-Saharan Africa. Some breeds of cattle are genetically more resistant to the pathogenic effects of trypanosome infection. We measured B-cell activation and the quantity and isotype of antibody produced at the cellular level in six trypanotolerant N'Dama and five trypanosusceptible Boran cattle. The frequencies of spleen cells secreting total and parasite-specific IgM and IgG were measured prior to and 16, 28, and 35 days after a primary challenge withTrypanosoma congolense.Boran cattle had higher frequencies of splenic cells secreting IgM specific for trypanosome-derived variable surface glycoprotein (VSG), cysteine protease (congopain, CP), and heat shock protein (hsp70/BiP) and the nonparasite antigen, ovalbumin, than did N'Dama cattle. In contrast, the number of VSG-specific IgG-secreting cells was significantly greater in N'Dama than in Boran cattle. During infection, low titers of anti-VSG IgM were detected transiently in the serum of all animals. However, N'Dama had significantly more VSG-specific IgG in blood than Boran during infection. The peripheral blood mononuclear cell population of N'Dama cattle contained a higher percentage of surface IgM-positive B-cells prior to and throughout infection than were found in the blood of Boran. In addition, during infection N'Dama cattle had more circulating lymphocytes that could be activatedin vitroto undergo differentiation into IgM- and IgG-secreting cells. These findings demonstrate differences in the frequency of trypanosome-specific antibody-secreting cells in the spleen and in the activation state of B-cells in the blood between N'Dama and Boran cattle during a primary infection withT. congolense

Guidance on the assessment criteria for studies evaluating the effectiveness of stunning interventions regarding animal protection at the time of killing

This guidance defines the assessment process and the criteria that will be applied by the Animal Health and Welfare Panel to studies on known new or modified legal stunning interventions to determine their suitability for further assessment. The criteria that need to be fulfilled are eligibility criteria, reporting quality criteria and methodological quality criteria. The eligibility criteria are based upon the legislation and previously published scientific data. They focus on the intervention and the outcomes of interest, i.e. immediate onset of unconsciousness and insensibility or absence of avoidable pain, distress and suffering until the loss of consciousness and sensibility, and duration of the unconsciousness and insensibility (until death). If a study fulfils the eligibility criteria, it will be assessed regarding a set of reporting quality criteria that are based on the REFLECT and the STROBE statements. As a final step in this first assessment phase, the methodological quality of the submitted study will be assessed. If the criteria regarding eligibility, reporting quality and methodological quality are fulfilled, a full assessment of the animal welfare implications of the proposed alternative stunning intervention, including both pre-stunning and stunning phases, and an evaluation of the quality, strength and external validity of the evidence presented would be carried out at the next level of the assessment. In the case that the criteria regarding eligibility and reporting quality and methodological quality are not fulfilled, the assessment report of the panel will highlight the shortcomings and indicate where improvements are required before the study can be assessed further. In addition to the assessment criteria, the guidance also specifies general aspects applicable to studies on stunning interventions that should be considered when studying the effectiveness of stunning interventions