Immunoglobulin isotype response of Fasciola spp.infected sheep and cattle to defined Fasciola spp. antigens

Following the general introduction, chapter one, the thesis is divided into four chapters, covering literature review, materials and methods, results and discussion and conclusion.Fasciolosis is a liver fluke disease, caused by Fasciola hepatica in temperate regions and high altitude areas of the tropics and subtropics and by Fasciola gigantica, which is restricted to the tropics and subtropics. Liver flukes have a wide range of definitive hosts, including man and in particular domestic ruminants, but the various hosts are known to differ greatly in their resistance to infection with these parasites. For example sheep are considered susceptible to challenge infection while cattle develop resistance.F. hepatica secretes the enzyme cathepsin-Ll protease (Fh-cathepsin) which has a molecular weight (MWt) of 27 kDa. It is considered to have a functional role in parasite evasion of the host immune response, through cleavage of host immunoglobulin. The enzyme, glutathione s-transferase (GST) is of 27.8-29 kDa MWt, is also secreted by F. hepatica (Fh-GST) and is thought to be involved in the detoxification of exogenous (xenobiotic) and endogenous derived toxic compounds. Both enzymes form part of the fluke excretory/secretory (E/S) products and are of interest as they are considered as vaccine candidates against fasciolosis.This study investigated the immunoglobulin isotype responses of sheep and cattle, chronically infected with F. hepatica and F. gigantica, to defined the fluke antigens (F. hepatica E/S products (Fh-E/S) or F. gigantica E/S products (Fg-E/S), Fhxii cathepsin and Fh-GST). It was decided to study the immune response in chronically infected animals since immunity is considered to play a potentially more important role in chronic infection, than in acute infection, which is characterised by the death of the animal through anaemia and blood loss caused by the migrating flukes. Serum and faecal samples were collected weekly while the severity of the infections were defined using clinical, parasitological, haematological, biochemical and pathological parameters.Serum and faecal antibody (total Ig, IgGb IgM, IgG2 and IgA) responses to 24- 48 hour Fh-E/S and Fg-E/S, adult Fh-cathepsin and adult Fh-GST were determined by indirect Enzyme-linked Immunosorbent Assay (ELISA). The antigen recognition profile of the Fasciola spp. infected sheep and cattle to Fh-E/S and Fg-E/S was examined by sequential Western blotting.The general clinical and pathological pattern, combined with the parasitological, biochemical and haematological data confirmed that, in the main, the sheep and cattle were suffering from chronic fasciolosis. Lesions were most severe in sheep culled 11 to 20 weeks post infection (wpi) and calves with single infection, but culled 12 wpi, and those calves with challenge infection. Overall, the calves had very light infection and as a result they displayed a less severe chronic fasciolosis than that observed in sheep.A reduction in serum glucose levels was detected in F. hepatica and F. gigantica infected sheep from 3 wpi, especially in severely infected sheep. In contrast, there was an increase in serum P-hydroxybutyrate (P-HOB) levels in infected sheep, from about 6 to 15 wpi. And the extent of the rise in P-HOB levels was associated with the severity of the infection There was no serum glucose or P-HOB changes observed in infected calves. This was considered to be due to the very light fluke infection. Thus, it would appear from the serum glucose and P-HOB levels, that fasciolosis leads to energy deficiency (low glucose) and ketosis (increased b-HOB) especially noticeable in more heavily infected animals.There was an early (2-3 wpi) total Ig response to Fh-E/S and Fg-E/S, Fhcathepsin and Fh-GST in both F. hepatica infected sheep and cattle. Although there was an early (2-3 wpi) total Ig response to Fh-E/S and Fg-E/S, and Fh-GST by F. gigantica infected animals, there was a slight delay (7 wpi) noted in the response to Fh-cathepsin. The pattern of the IgGj response of cattle and sheep to these defined fluke antigens was similar to that of total Ig. In fact the serum isotype response was predominantly IgG]. The IgM response to Fh-E/S and Fg-E/S, Fh-cathepsin and Fh-GST was early in both species. In cattle the IgG2 and IgA responses to Fh-E/S and Fg-E/S were late and more pronounced (11 wpi and 19 wpi respectively) in contrast to sheep (2 wpi for both isotypes). The serum IgG2 and IgA isotype responses to Fh-cathepsin and Fh-GST followed the same pattern in cattle, however in sheep, responses to Fh-cathepsin was much less marked and a response to Fh-GST was not detected. A rise in total Ig and IgG, responses to Fh-E/S and Fg-E/S and Fh-cathepsin were detected following challenge infection in calves, but there was no increase in the response to Fh-GST. The dominance of the IgGj response in Fasciola spp. infected sheep and cattle suggests an associated Th2 response in both species. The late IgG2 response in cattle may suggest late Th, involvement in bovine cellular responses to Fh-E/S and Fg-E/S products.The detection of serum antibody responses to Fh-cathepsin and Fh-GST in F. gigantica infected sheep and cattle confirmed antigenic cross-reactivity. However this xiv cross-reactivity may be only partial, as suggested by the late total Ig and IgG, response to Fh-cathepsin by F. gigantica infected sheep and cattle. Comparison of the isotype responses to Fh-E/S and Fg-E/S products, Fh-cathepsin and Fh-GST suggest that there is very little difference between the response to Fh-E/S, Fg-E/S and Fh-cathepsin, however, there was a relatively poor response to Fh-GST in both sheep and cattle.Western blot analysis ofF. hepatica infected sheep serum identified antigens of 14 kDa from 2 wpi and 54, 79 and 134 kDa MW recognised later. In F. gigantica infected sheep antigens of 14, 88 and 152 kDa were identified from 7-9 wpi. There was a clear shift, at patency, in the antigen recognition pattern of cattle from higher (134 kDa for F. gigantica infection and 142 kDa for F. hepatica infection) to lower (60 kDa for both parasite species) but there was no clear antigenic shift observed in sheep. None of these antigenic molecules represented Fh-cathepsin (27 kDa MWt) or Fh-GST (27.9- 29 kDa MWt) used as antigens in the ELISA assay. The lower protein concentration of the E/S products used in this assay is, one possible reason for failure to detect these molecules.There was no faecal antibody response detected in cattle to any of the three defined antigens. This might have been due either to the light infections observed in cattle or to the larger volume of faecal material produced by cattle (i.e. dilution). There was an early (2 wpi) faecal total Ig response to Fh-E/S and Fg-E/S, Fh-cathepsin and Fh-GST in F. hepatica and F. gigantica infected sheep. In fact there was no difference in faecal antibody responses to the different antigens by either F. hepatica or F. gigantica infected sheep. The isotype response was mainly IgA while a slight IgG2 response could be detected in F. hepatica infected sheep. The total Ig and IgA responses XV to all three defined antigens in F. hepatica sheep was biphasic, in F. gigantica infection however the phases were less defined. The 1st phase (about 2-10 wpi) is considered a response to juvenile flukes antigens after oral infection and the second peak (13-17 wpi) is considered to be in response to antigens released by adult flukes in the bile duct.This study indicates that in serum, IgG( isotype responses predominate and in faeces IgA isotype responses to the three defined antigens predominate, suggesting preferentially stimulation of the Th2 T-cell subset in sheep and cattle fasciolosis. The late IgG2 response to F. hepatica and F. gigantica E/S and Fh-cathepsin in cattle may indicate a delayed Th, stimulation.In order to elucidate the different immune mechanisms operating in infected sheep and cattle there is need for more work on the cellular responses to Fasciola spp. as these may be related to resistance. There was antigenic shift in cattle at patency but not in sheep. The 60 kDa molecule being recognised by cattle after the antigenic shift. It is possible that this factor may be related to acquisition of resistance by cattle to secondary Fasciola spp. infectionFinally the findings relating to the glucose and b- HOB levels in infected sheep may prove helpful in experiments involved in the interaction between fasciolosis and the nutritional levels of Fasciola spp infected animals, of particular importance in young growing animals

Perceptions and acceptability of piloted Taenia solium control and elimination interventions in two endemic communities in eastern Zambia

Infections with Taenia solium cause significant public health and economic losses worldwide. Despite effective control tools, long‐term sustained control/elimination of the parasite has not been demonstrated to date. Success of intervention programs is dependent on their acceptability to local communities. Focus group discussions (FGDs) and questionnaires (QS) were conducted in two study communities in eastern Zambia to assess local perceptions and acceptance of two piloted intervention strategies: one targeting pigs only (‘control’ study arm), and one integrated human‐ and pig‐based (‘elimination’) strategy. QS (n = 227) captured data regarding participation in project activities, knowledge and perceptions of T. solium and of the interventional drugs used in the study. FGDs (n = 18) discussed perceived advantages and disadvantages of the interventions and of the project's delivery and value. QS data revealed 67% of respondents participated in at least one educational activity, and 80% correctly identified at least one disease targeted by the education. All elimination study arm respondents (n = 113) had taken the human treatment, and 98% intended to do so next time. Most (70%) indicated willingness to pay for future treatments (median 0.20 USD per dose). Of pig‐owning respondents, 11/12 (92%) had allowed their pigs to be treated/vaccinated and all intended to do so again next time. Four pig owners indicated willingness to pay 0.10–0.50 USD per dose of treatment or vaccine. FGD feedback revealed positive perceptions of interventions; people reported improved health in themselves and their pigs, and fewer cysticerci in pork. Latrine use, hand washing, meat inspection and proper cooking of pork had reportedly increased since the program's inception. Preliminary assessment indicates that the piloted intervention methods are generally acceptable to the communities. The reported willingness of many respondents to pay for the medications would contribute to the feasibility of long‐term, government‐led T. solium intervention programs in future

Effects of ‘The Vicious Worm’ educational tool on Taenia solium knowledge retention in Zambian primary school students after one year

Background: Taenia solium is a neglected zoonotic parasite endemic throughout many low-income countries worldwide, including Zambia, where it causes human and pig diseases with high health and socioeconomic burdens. Lack of knowledge is a recognized risk factor, and consequently targeted health educational programs can decrease parasite transmission and disease occurrence in endemic areas. Preliminary assessment of the computer-based education program The Vicious Worm' in rural areas of eastern Zambia indicated that it was effective at increasing knowledge of T. solium in primary school students. The aim of this study was to evaluate the impact of The Vicious Worm' on knowledge retention by re-assessing the same primary school students one year after the initial education workshops. Methodology/Principal findings: Follow-up questionnaires were administered in the original three primary schools in eastern Zambia in 2017, 12 months after the original workshops. In total, 86 pupils participated in the follow-up sessions, representing 87% of the initial workshop respondents. Knowledge of T. solium at follow-up' was significantly higher than at the initial pre' questionnaire administered during the Vicious Worm workshop that took place one year earlier. While some specifics of the parasite's life cycle were not completely understood, the key messages for disease prevention, such as the importance of hand washing and properly cooking pork, remained well understood by the students, even one year later. Conclusions/Significance: Results of this study indicate that The Vicious Worm' may be an effective tool for both short- and long-term T. solium education of rural primary school students in Zambia. Inclusion of educational workshops using The Vicious Worm' could be recommended for integrated cysticercosis control/elimination programs in sub-Saharan Africa, particularly if the content is simplified to focus on the key messages for prevention of disease transmission. Author summary The zoonotic parasite Taenia solium, commonly known as the pork tapeworm, causes substantial public health and economic losses worldwide. It is commonly found in low-income countries where pigs are raised in areas of poor sanitation, including Zambia. The links between the parasite and its different disease forms in humans and pigs are not very well known, and ignorance of the parasite is a known risk factor for infection. Health education can significantly increase knowledge and awareness of the parasite and can inspire behavioral change that reduces disease transmission. The Vicious Worm' is a computer-based program designed to provide T. solium education in a fun and interactive way. We conducted educational workshops in three primary schools in rural areas of eastern Zambia, and preliminary assessment indicated that the Vicious Worm' educational content significantly improved students' knowledge of T. solium. We also conducted follow-up studies in the same students one year later, and discovered that the students' knowledge was still significantly higher than at baseline. We conclude that The Vicious Worm' may be a useful educational component to enable targeting of school students, and would recommend its inclusion in integrated T. solium control programs in future