Research Article 2020 iMedPub Journals Stroke Research and Therapy www.imedpub.com Vol.4 No.2:2 Experimental Production of Anti-membrane Reymick Okwong-Oketch1, Julius Nsubuga2, Peter Bound Variable Surface Glycoglycoprotein Ayebare2, Zachary Nsadha2, (Anti-VSGm) Antibodies forin vitro and in George William Lubega 2 and situ Immunostaining of Pathogenic African Ann Nanteza2* Abstract Background: The variant surface glycoprotein (VSG) of the African trypanosomes 1College of Humanities and Socio- is the major membrane protein of the plasma membrane of the bloodstream sciences, Makerere University, Kampala, stage of the parasite. African trypanosomiasis (sleeping sickness in humans and Uganda nagana in animals) is caused by the systemic infection of the host by several sub- species of the extracellular haemoflagellate protozoa under genus Trypanosoma. As a defense barrier against the host immune response, the entire surface of the 2College of Veterinary Medicine, Animal bloodstream form of trypanosome is covered with densely packed molecules of Resources and Biosecurity, Makerere VSG that determines the antigenic phenotype of the parasite. Variant surface University, Kampala, Uganda glycoprotein has a C-terminal domain that is highly conserved in various species of trypanosomes. Methods: The membrane bound VSG (VSGm) protein was prepared without *Corresponding author: Ann Nanteza, denaturing the homologous region and by including numerous variable antigen College of Veterinary Medicine, Animal types from Trypanosoma brucei brucei parasites. The purified VSGm native Resources and Biosecurity, Makerere trypanosome protein was used to produce anti-VSGm immune sera in rabbits. The University, Kampala, Uganda, E-mail: indirect immunofluorescence assay (IFA) was used to detect trypanosomes from [email protected], Tel: +256 mice blood, artificial culture media and cattle histological sections. 772443584 Results: The resultant immune sera were able to detect different strains and species of African trypanosomes from in vivo and in situ sources after immunostaining. Anti-VSGm antibodies also demonstrated a unique property to Citation: Okwong-Oketch R, Nsubuga J, locate trypanosomes within the histological tissues even after the trypanosome’s Ayebare P, Nsadha Z, Lubega GW, et al. (2020) morphology had been distorted. Experimental Production of Anti-membrane Bound Variable Surface Glycoglycoprotein Conclusion: The produced immune sera can be utilized for immunohistochemistry (anti-VSGm) Antibodies for in vitro and in Trypanosoma to detect species in various fluids and tissues. situ Immunostaining of Pathogenic African Keywords: Trypanosome; Variable surface glycoprotein; Immunostaining; Antibodies; Stroke. Res Ther Vol.4 No.2. DOI: 10.36648/ Bloodstream stroke.4.2.2 Received: September 11, 2020; Accepted: October 05, 2020; Published: October 12, 2020 Introduction protein content and numbers of lymphocytes are found in the cerebrospinal fluid (CSF) [5]. African trypanosomiasis results from systemic infection of the mammalian host by several sub-species of the extracellular The drugs widely used in the treatment of second-stage hemoflagellate of the genus Trypanosoma. The parasites are HAT are highly toxic [6,7], therefore, both diagnosis and staging mainly transmitted by a bite of the blood-sucking tsetse fly of of the disease must be highly accurate as a correct diagnosis the genus Glossina [1], and after the initial skin infection, they is beneficial for both infected individuals and the community multiply extracellularly [2] by binary fission in the bloodstream, [8]. Diagnosis of trypanosomes follows a three-step pathway lymph and interstitial fluids [3], then spread via the lymph (screening, diagnostic confirmation and staging). Diagnostic and blood circulation within the host [4]. Human African confirmation mostly relies on identification of trypanosomes trypanosomiasis (HAT) in divided into an early hemolymphatic in the blood, lymph nodes or CSF. However, the standard stage, in which the parasites are confined to the blood and lymph, parasitological techniques available miss up to 30% of patients and a late encephalitic stage, in which T. brucei and increased [9]. Staging allows classification of HAT into the hemolymphatic © Under License of Creative Commons Attribution 3.0 License | This article is available in: https://stroke.imedpub.com/ 1 Stroke ResearchARCHIVOS and DE TherapyMEDICINA 2020 ISSN 1698-9465 Vol.4 No.2:2 or meningo-encephalitic stage but because of the absence of Experimental animals and trypanosome stocks reliable blood tests able to detect central nervous system (CNS) All the experimental animals were purchased from the invasion by the parasite, staging relies on the CSF examination COVAB, Small Animal Laboratory Experimental Unit, Makerere [8]. According to Kennedy [10] there is no consensusas to what University,Kampala, Uganda. Twelve (12) weeks old Albino exactly defines a biologically CNS disease or what specific CSF rats and four weeks old Albino mice were used to propagate findings should justify drug therapy for late-stage involvement trypanosomes. Fourteen [14] weeksold New Zealand White yet diagnosis of the CNS involvement is crucial in order to give an rabbits were used to produce anti-VSG immune sera following appropriate treatment regimen. their immunization by the purified VSGm native trypanosome Trypanosomes have a complex antigenic structure and protein. All control animals were sex and age-matched with elicit the production of a large spectrum of antibodies. Variant test animals in the immunization protocol. Trypanosome surface glycoprotein (VSG) is specific to trypanosomes thus anti- stabilates made from T. b. brucei UTRO 010291B, obtained from VSG antibodies that are able to stain various strains and/or species Molecular Biology Laboratory, Makerere University, Kampala, of trypanosomes would be very significant in mass screening and Uganda, courtesy of Prof. Enock Matovu were used for parasite staging of human trypanosomiasis. Such antibodies would for propagation leading to VSG protein extraction. Frozen stabilates example be coated on plates and be used for mass screening, had been prepared as described by [14]. detecting recently released VSG or trypanosomes in blood. This could reduce on incidences of false positives and false negatives Propagation of trypanosoma brucei brucei utro common in the card antigen test for Trypanosoma brucei 010291b in mice gambiense (CATT/T. b. gambiense) [11], but also reduce on time All experimental infections for parasite propagations were lag between sampling and examination to avoid immobilization made after at least one mouse passage from frozen stabilate and subsequent lysis of trypanosomes in the samples. to revitalize the trypanosomes. Mice were always infected by Microscopic examination of lymph node aspirate, blood intraperitoneal injections of 1 × 104 trypanosomes in 200 µl of or CSF provides direct evidence for trypanosome infections. Phosphate Saline Glucose (PSG) buffer. The trypanosomes were However, most of the available parasite detection methods would propagated in three [3] mice and parasitaemia monitored daily not detect the parasites if their number is below the detection by wet preparations from tail blood and its levels determined by limit of 100 trypanosomes/ml [12]. Anti-VSG antibodies, however, Matching method [15]. can detect even small amounts of the VSG protein in released or soluble form, thus could be used to supplement the current Purification oftrypanosoma brucei brucei parasites methods used in microscopic examination, especially of CSF from whole blood obtained by lumbar puncture during disease staging in HAT. This This was done by chromatography using diethylaminoethyl could allow detecting VSG protein in CSF even after ten minutes (DEAE)-cellulose (Sigma Aldrich, Germany); regenerated of sample collection in which trypanosomes begin to lyse [8], according to manufacturer’s protocol and packed into a column but also promote sensitivity of trypanosome detection in CSF (50 ml syringe) using the gravity flow, then equilibrated using at less time, unlike the double centrifugation method which is PSG buffer until the effluent was pH 8. The rats were sacrificed time consuming and requires two centrifuges [8,13]. This study when the parasitemia was high (108.2-108.4 trypanosomes/ml of produced anti-VSG immune sera in rabbits that could be used to blood) and blood was drawn by cardiac puncture into 8 ml EDTA stain heterologous strains and species of African trypanosomes in vacutainer tubes. The EDTA blood was loaded into the column vitro and in situ. and the components allowed to flow into the DEAE cellulose. Then 1x PSG (pH 8) buffer was continuously added to the column Methods until all the trypanosomes had passed through it as determined Ethics statement and considerations by checking the effluent microscopically at intervals. An aliquot (10 µl) was used to count the number of trypanosomes in the The study was carried out in accordance with the recommendations effluent using a Neubauer haemocytometer. The effluent was in the Standard Operating Procedures (SOPs) of experimental then centrifuged at 3500 revolutions per minute (rpm) for twenty animal care of the College of Veterinary Medicine, Animal minutes, the supernatant was poured off and the mass/weight Resources and Biosecurity (COVAB), Makerere Universit and of the pellet determined. The pellets were snapping frozen and adequate consideration of the 3R's (Replacement