Molecular biology and immunology of the 60-kDa stress protein (65-kDa antigen) of Mycobacterium paratuberculosis

M. paratuberculosis infection in sheep causes a chronic granulomatous enteritis, characterised by a massive cellular infiltration of macrophages, epithelioid cells and lymphocytes. Like other mycobacterial infections the predominant immune response is cell-mediated and this response centres on a complex interaction between T cells and macrophages infected with M. paratuberculosis organisms. T cells have been identified as the predominant inducers of the cell mediated immune response to mycobacteria. Stress proteins have been shown to be immunodominant antigens in immune responses to other pathogenic mycobacteria. T cells reactive to stress proteins have been isolated from infected animals. For these reasons, the 60-kDa stress protein of M. paratuberculosis was chosen for the investigation of the immune response to M. paratuberculosis infection in sheep.In this thesis the DNA encoding the 60-kDa stress protein of M. paratuberculosis was amplified by PCR, sequenced and the ORF of this protein expressed as a fusion protein with glutathione S-transferase. Recombinant M. paratuberculosis 60-kDa stress protein was obtained by cleavage of the fusion protein with the proteolytic enzyme thrombin.Recombinant M. paratuberculosis 60-kDa stress protein was used to generate polyclonal T and B cell responses in sheep which were assessed by in vitro proliferation and enzyme-linked immunosorbent assays, respectively. In addition, the recombinant protein was used to generate a monoclonal antibody. The assays and reagents generated in this thesis were subsequently used to investigate the immune response to M. paratuberculosis 60-kDa stress protein in ovine paratuberculosis.This investigation provides preliminary evidence that paratuberculosis-infected animals have T cells that recognise and are capable of responding by proliferation to M. paratuberculosis 60-kDa stress protein in vitro, and that antibodies to this protein could be detected in sera from these animals. Furthermore, M. paratuberculosis 60-kDa stress protein was detected in tissues from infected animals by immunocytochemical analysis. These preliminary findings support a role for M. paratuberculosis 60-kDa stress protein in the ovine immune response to M. paratuberculosis infection

Implementation of a practical and effective pilot intervention against transmission of Taenia solium by pigs in the Banke district of Nepal.

Taenia solium is a zoonotic cestode parasite which causes human neurocysticercosis. Pigs transmit the parasite by acting as the intermediate host. An intervention was implemented to control transmission of T. solium by pigs in Dalit communities of Banke District, Nepal. Every 3 months, pigs were vaccinated with the TSOL18 recombinant vaccine (Cysvax, IIL, India)) and, at the same time, given an oral treatment with 30mg/kg oxfendazole (Paranthic 10% MCI, Morocco). The prevalence of porcine cysticercosis was determined in both an intervention area as well as a similar no intervention control area, among randomly selected, slaughter-age pigs. Post mortem assessments were undertaken both at the start and at the end of the intervention. Participants conducting the post mortem assessments were blinded as to the source of the animals being assessed. At the start of the intervention the prevalence of porcine cysticercosis was 23.6% and 34.5% in the control and intervention areas, respectively. Following the intervention, the prevalence of cysticercosis in pigs from the control area was 16.7% (no significant change), whereas no infection was detected after complete slicing of all muscle tissue and brain in animals from the intervention area (P = 0.004). These findings are discussed in relation to the feasibility and sustainability of T. solium control. The 3-monthly vaccination and drug treatment intervention in pigs used here is suggested as an effective and practical method for reducing T. solium transmission by pigs. The results suggest that applying the intervention over a period of years may ultimately reduce the number of tapeworm carriers and thereby the incidence of NCC

Control trial of porcine cysticercosis in Uganda using a combination of the TSOL18 vaccination and oxfendazole

Neurocysticercosis caused by Taenia solium when the parasite lodges in the central nervous system, is an important cause of human seizures and mortality in sub-Saharan Africa. The parasite is prevalent in many regions of Uganda. Pigs are intermediate hosts for T. solium , and we evaluated a T. solium control program in pigs, involving vaccination of pigs with the TSOL18 vaccine and treatment with oxfendazole

Accurate diagnosis of lesions suspected of being caused by Taenia solium in body organs of pigs with naturally acquired porcine cysticercosis.

The definitive method for diagnosis of porcine cysticercosis is the detection of cysticerci at necropsy. Cysts are typically located in the striated muscle and brain. Until recently Taenia solium cysticerci have not been definitively identified in other tissue locations, despite several comprehensive investigations having been undertaken which included investigation of body organs other than muscle and brain. Recently a study conducted in Zambia reported 27% infection with T. solium in the liver of pigs with naturally acquired porcine cysticercosis, as well as some T. solium infection in the lungs and spleen of some animals. We investigated the cause of lesions in sites other than the muscle or brain in a total of 157 pigs from T. solium endemic regions of Uganda and Nepal which were subjected to extensive investigations at necropsy. Lesions which had the potential to be caused by T. solium were characterised by macroscopic and microscopic examination, histology as well as DNA characterisation by PCR-RFLP and sequencing. Lesions were confirmed as being caused by Taenia hydatigena (both viable and non-viable), by T. asiatica and Echinococcus granulosus (in Nepal) and nematode infections. No T. solium-related lesions or cysticerci were identified in any tissue other than muscle and brain. It is recommended that future evaluations of porcine cysticercosis in aberrant tissue locations include DNA analyses that take appropriate care to avoid the possibility of contamination of tissue specimens with DNA from a different tissue location or a different animal. The use of appropriate control samples to confirm the absence of cross-sample contamination is also recommended

TSOL18 vaccine and oxfendazole for control of Taenia solium cysticercosis in pigs: A field trial in endemic areas of Tanzania.

A field trial was conducted in Tanzania to determine the effectiveness of TSOL18 vaccine used concurrently with oxfendazole (OFZ), and of OFZ alone, on T. solium cysticercosis determined by organ and half carcase dissection of slaughter age pigs. This study followed a quasi-experimental group design. Suitable trial sites were randomly allocated to either treatment group T1 (OFZ treatment alone [30mg/kg, Paranthic 10%]) or T2 (TSOL18 [1ml, Cysvax] plus OFZ). Three 4-monthly treatments were administered to eligible pigs. A random selection of pigs were necropsied at baseline and at endline, 2-3.5 months after the final treatment. Additionally, untreated pigs from T1 and T2 areas were necropsied at endline to provide contemporaneous comparisons with T1 and T2 pigs. Baseline prevalence of viable T. solium cysticerci for T1 was 25.5% (Exact 95% CI: 13.9, 40.3; n = 12/47), and for T2 was 12.0% (CI: 6.4, 20.0; n = 12/100). At endline, prevalence was 2.8% for T1 (CI: 0.1, 14.5, n = 1/36) and 0% for T2 (CI: 0, 4.7, n = 0/77). Among untreated pigs, three had viable cysticerci, one from T1 area (12.5%, CI: 0.3, 52.7; n = 1/8) and two from T2 area (5.7%, CI: 0.7, 19.2, n = 2/35). Fisher's exact test showed significant changes in prevalence from baseline to endline in both groups (T1: p = 0.005, T2: p = 0.001). Firth's penalized Maximum Likelihood method suggested the changes were not significant relative to their controls (T1: p = 0.245, T2: p = 0.076). These findings showed a significant reduction in the prevalence of viable cysticerci from baseline to endline after both interventions. However, the changes could not be definitively attributed to the interventions due, in part, to small numbers of control pigs. Concurrent administration of the TSOL18 and OFZ cleared infection among assessed pigs whereas infection remained after treatment with OFZ only. Further studies including larger sample sizes would be required for more definitive conclusions. A One Health approach is recommended for rapid and sustainable impact

Taenia solium from a community perspective : preliminary costing data in the Katete and Sinda districts in Eastern Zambia

The tapeworm Taenia solium is endemic in Zambia, however its socioeconomic cost is unknown. During a large-scale interventional study conducted in Zambia, baseline economic costs of human and porcine T. solium infections were measured. Questionnaire surveys were conducted within three neighbourhoods in Zambia's Eastern province in 2015 and 2016. A human health questionnaire, capturing costs of clinical symptoms commonly attributable to human cysticercosis and taeniasis, was conducted in randomly selected households (n = 267). All pig-keeping households were administered a pig socioeconomic questionnaire (n = 271) that captured pig demographic data, costs of pig-keeping, and economic losses from porcine cysticercosis. Of all respondents 62% had reportedly experienced at least one of the surveyed symptoms. Seizure-like episodes were reported by 12%, severe chronic headaches by 36%, and vision problems by 23% of respondents. These complaints resulted in 147 health care consultations and 17 hospitalizations in the five years preceding the study, and an estimated productivity loss of 608 working days per year. Of all pigs 69% were bought within villages. Nearly all adult pigs were sold to local traders, and tongue palpation for detection of cysticerci was commonly performed. Reportedly, 95% of pig owners could not sell tongue-positive pigs, while infected pigs fetched only 45% of the normal sale value. These preliminary costing data indicate that human and porcine T. solium infections substantially impact endemic areas of Eastern Zambia. A full socioeconomic burden assessment may enable improved T. solium management in sub-Saharan Africa