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

Background 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. Methods The study was conducted in two districts of Eastern Uganda involving the rural village communities of Bukedea (intervention area) and Kumi (control area) during 2016–2017. Seven hundred and thirty-four households were enrolled in the study. Pigs in the intervention area received intramuscular immunizations with TSOL18 (Cysvax™) and an oral medication with 30 mg/kg oxfendazole (Paranthic™) at approximately 3-monthly intervals for 18 months. Porcine cysticercosis was evaluated by post-mortem examination. At the beginning of the study, 111 pigs were examined. In an interim evaluation in the intervention area, 55 pigs were evaluated 12 months after starting the project. At the end of the study approximately 3 months after the final intervention, 55 pigs from the intervention area and 56 pigs from the control area were evaluated. Results The prevalence of porcine cysticercosis for the two sites was 16.2% at the beginning of the study (17.2% in the intervention area and 15.1% in the control area) with no statistically significant difference (P = 0.759) between the two study sites. Among the 110 animals assessed from the intervention site (55 at the interim evaluation and 55 at the final evaluation), no pig with viable T. solium cysts was found. There was a statistically significant difference between the prevalence at baseline (17.2%) and at the end of the study (0%) in the intervention area (P = 0.001) and a statistically significant difference between the intervention (0%) and control areas (5.4%) (P = 0.041) at the end of the study. Conclusions Three-monthly concurrent vaccination of pigs with the TSOL18 vaccine and medication with oxfendazole eliminated T. solium transmission by the animals involved in the study. Application of vaccination with medication in pigs has the potential to reduce transmission of T. solium in Uganda and other endemic countries.

there is substantial evidence for a high prevalence of porcine cysticercosis in many parts of Uganda [4,[6][7][8], there is very little data published about the prevalence of human neurocysticercosis in that country. Winkler and Richter [9] cite Winkler unpublished data as indicating a neurocysticercosis prevalence rate of 10% in persons with epilepsy living in rural areas of Uganda. Investigations in Uganda of subcutaneous lesions in onchocerciasis patients who suffered epilepsy were found in many cases to be caused by T. solium [10]. Alarakol and colleagues [11] found that up to 19.6% of epileptic patients in Uganda were serological positive for anti-T. solium antibodies. It seems likely that neurocysticercosis exacts a significant toll on human morbidity in Uganda, but that it is underreported.
There has been increasing interest in investigating the potential for control of transmission of T. solium by pigs through the combined use of the TSOL18 vaccine together with treatment at the time of vaccination with oxfendazole in order to eliminate parasites that may have established prior to vaccination [12,13]. Field trials of this strategy have been successful in eliminating T. solium infection in pigs in Cameroon [14], Nepal [15] and Tanzania [16]. Programs incorporating pig vaccination and treatment, as well as treatment of the human population with taeniacide, have also been effective in eliminating T. solium transmission by pigs in a northern area of Peru [17] and in Zambia [18].
To date, there has been little attention paid to investigating potential strategies to reduce the transmission of T. solium in Uganda that could reduce the incidence of human neurocysticercosis. In order to assess the effectiveness of a vaccination strategy for reducing T. solium transmission through pigs in Uganda, we undertook a field evaluation of the TSOL18 vaccine in the Bukedea and Kumi districts of Eastern Uganda.

Study design
The study was conducted in rural village communities in Bukedea and Kumi districts of Eastern Uganda. Two distinct areas were involved, one a control area and the other an intervention area where pigs received concurrent vaccination with TSOL18 and medication with oxfendazole. Prior to undertaking baseline assessments of the prevalence of porcine cysticercosis, site surveys identified Bukedea and Kumi as suitable study areas, where pig owners were willing to consent to their pigs participating in the study. The study was undertaken in animals belonging to farmers who agreed to cooperate in the project. The owners and pigs were representative of the population of pigs of the study areas. Immediately prior to the trial beginning, healthy slaughter-age and weight pigs were randomly selected from pig owning households in the two study areas and subjected to post-mortem examination to determine the prevalence of porcine cysticercosis.

Selection of study animals
The eligibility criteria for animals that were enrolled in the study were pigs > 8 weeks of age, not heavily pregnant and not clinically ill. In the treatment area, animals that were destined for slaughter within three weeks were excluded in compliance with the withholding period for the oxfendazole formulation that was used [19].

Vaccination and pig medication
The day of first treatment administration to pigs was defined as Day 0. The intervention study site was visited at intervals of approximately 3 months and all pigs that met the inclusion criteria were enrolled continuously into the study, ear tagged, weighed, vaccinated and treated with oxfendazole. Animal weight in kilograms was estimated using a measuring tape (inches) and the formula [Girth 2 × length/400]/2.2. The dose for oxfendazole (3 ml/10 kg Paranthic ™ 10%, MCI Sante Animale, Morocco) was calculated according to the animal weight (30 mg/kg body weight) and was applied per os. Concurrently, 1 ml TSOL18 vaccine (150 µg TSOL18 in mineral oil adjuvant; Cysvax ™ , Indian Immunologicals Limited, India) was administered intramuscularly in the left side of the neck behind the base of the ear, prior to release of the animal. TSOL18 vaccine and oxfendazole were administered on Days 0 to 15 (first intervention), Days 106 to 118 (second intervention), Days 209 to 220 (third intervention), Days 308 to 318 (fourth intervention), Days 404 to 419 (fifth intervention), and Days 505 to 518 (sixth intervention).

Assessment of pig infections
Infection with T. solium was determined at necropsy. The study was designed to be able to identify an 80% reduction in the prevalence of porcine cysticercosis in slaughter-age and weight pigs.
Sample size calculations were undertaken using a one-sided likelihood ratio test at the 5% significance level using SAS 9.3 (SAS Institute, Cary North Carolina, USA) with the TWOSAMPLEFREQ command in the PROC POWER procedure. Assuming an initial prevalence of infection of 20%, sample sizes of 55 animals were required in each area at the start and end of the trial in order to meet the desired statistical power. As a baseline, on Days -22 to -13, 58 slaughter-age pigs from the intervention area and 53 pigs from the control area were assessed. Approximately 3 months after the fourth intervention (on days 386-391) an interim evaluation of infection levels was undertaken in 55 animals that had received treatments on at least two occasions selected randomly from the intervention area. A final evaluation was made approximately 2-2.5 months after the final intervention involving a further 55 slaughter-age animals selected randomly from the intervention area, and 56 slaughter-age animals selected randomly from the control area on Days 572-580.
Post-mortem procedures were similar to those described by Sah et al. [20] and Poudel et al. [15]. The animals were transported to the National Animal Disease Diagnostics and Epidemiology Centre (NADDEC) Laboratories in Entebbe where they were euthanized by captive bolt pistol by experienced staff. The viscera were removed and the heart, liver, both kidneys and the full diaphragm retained in numbered containers. The carcase was skinned and divided cranio-caudally along the spine. The organs and the carcase, including the complete head, were refrigerated overnight at 4 °C. The head was removed and the tongue, masticatory muscles (both right and left sides) and brain were removed and retained. The muscles from the carcase were dissected from the bones.

Examination for Taenia solium cysts
Except in cases of very heavy infection, all the retained organs and muscles of the right side of the carcase were sliced by hand at intervals of approximately 3 mm and examined meticulously for the presence of T. solium cysticerci or other lesions. During the necropsies undertaken on 111 animals at the end of the trial, when no cysticerci were detected in the tongue, masticatory muscles, diaphragm, brain or muscles from the right hand side of the carcase, the muscles of the left hand side of the carcase were also sliced. Cysticerci were counted and recorded as viable when they appeared as translucent vesicles filled with transparent fluid and having a visible white scolex. Non-viable lesions were recorded separately in cases where vesicles were non-translucent, containing a dense white or yellowish fluid and having no scolex and in cases of fibrosed or calcified lesions. Examples of suspect, non-viable lesions detected in organs other than muscle that were not calcified were placed into RNAlater (Sigma) and investigated by PCR analysis of a fragment of the mitochondrial 12S rDNA gene using the restriction enzymes DdeI and HinfI or HpaI, as described by Gauci et al. [21]. In carcases that contained thousands of cysts, all the heart, liver, kidneys, lungs, diaphragm, tongue, masticatory muscles and brain were sliced and counted and recorded as above. The remaining carcase musculature was weighed and representative samples from different muscle sites were selected representing approximately 1 kg. This sample was weighed accurately and then sliced and cysts counted as above and the number of cysts in the carcase muscles estimated from the total muscle weight.

Definition of a case of confirmed porcine cysticercosis
The definition of a confirmed case of cysticercosis which was adopted by Sah et al. [20] was also used here. An animal was determined to be a confirmed case of porcine cysticercosis if one or more viable T. solium cysticerci was found in the muscle and or the brain, or if more than one non-viable lesion was detected in the muscles and/or brain. Animals having only non-viable lesions in organs that are not typical locations for T. solium (eg the liver, lungs or kidneys), and which could not be confirmed as being T. solium by DNA analyses, were excluded.

Data analysis
Raw data was transcribed into pre-formatted Excel spreadsheets suitable for importation into the statistical software Genstat ® 18th edition (VSNi, registered in England and Wales). Results were summarised by treatment. Statistical analyses were undertaken to evaluate the effects of treatments on the prevalence of T. solium cysts at post-mortem examinations. Determination of the changes over a 24-month period in prevalence of T. solium cysts determined by carcass dissection of slaughter weight pigs in the intervention group were compared with the non-intervention control group. Prevalence of infection was determined before Day 0 and again after approximately 24 months. Results were compared within study treatment groups at baseline and end of study, using a two-sample binomial test. A generalised linear model with logit link function (logistic model) for binary data was used to compare results at baseline and endline between the groups and to provide standard error estimates and confidence intervals around prevalence figures (Genstat ® 18th edition). Prevalence data were based on cysts detected in the specified organs and muscles of the left-hand side of the carcase only. During endline necropsies, when no infection was detected in the organs or left side of the carcase, muscles of the right side were also sliced. Where any cysts were detected only in the rightside muscles, these data are presented descriptively, but were not included in the statistical comparisons.

Intervention
Seven hundred and thirty-four households were enrolled in the study. Vaccination and medication were administered to 7535 pigs and a total of 12,204 doses of Cysvax vaccine and medication were used during the six interventions. Trial staff recorded no adverse effects in pigs after vaccination and medication. Overall, the number of pigs vaccinated and treated at each intervention remained approximately the same, averaging 2034 pigs (Table 1). All the pigs that met the inclusion criteria were vaccinated, however pigs that did not meet the inclusion criteria were not recorded so the coverage could not be determined. The average weight pigs of at each intervention varied between 16.9 and 24.0 kg over the six interventions ( Table 1). The weight range was greater during the first three interventions (maximum 150 kg) compared with the last three interventions (maximum 95 kg).
The numbers of pigs which received 1, 2, 3, 4, 5, or 6 interventions during the trial is shown in Table 2. Forty-four percent of pigs had two or more vaccinations over the six interventions.
Numbers of pigs which received 1, 2, 3, 4, 5, or 6 interventions during the trial are shown in Table 2. Pigs would not have received all the treatments as there was a natural attrition due to sales and slaughter as the pigs reached the slaughter/sale weight.
The number of interventions received by each pig necropsied at the end of study post-mortems is shown in Table 3. Out of 55 pigs (29.1%) 16 had one intervention, 19 (34.6%) had two interventions, 13 (23.6%) had three interventions, three (5.5%) had four interventions, two (3.6%) had five interventions and two pigs (3.6%) had six interventions. The average weight of the intervention pigs was 25.4 ± 8.5 kg compared to 24.3 ± 7.8 kg for the control pigs, but the difference was not significantly different (P > 0.05).

Prevalence and intensity of porcine cysticercosis
The prevalence of pigs infected with viable cysticerci at the three different times that necropsies were undertaken during the trial is shown in Table 4. During the baseline post-mortem evaluations 10 out of 58 (17.2%) pigs were positive from the intervention area and eight out of 53 (15.1%) pigs positive from the control area. There was no significant difference between prevalence in the two areas (P = 0.759) using the logistic regression model. The overall prevalence of porcine cysticercosis was 16.2%. Of the 55 pigs necropsied from the intervention area during the interim evaluation, no animal was found to harbour viable T. solium infection. Similarly, at the end of the study none of the 56 pigs necropsied from the intervention area was found to have viable T. solium infection. In the control area five out of 56 pigs (8.9%) were found to have viable T. solium infection at endline, however, for two of these animals the cysts were only the right side of the carcase. At baseline the right side of the carcase was not evaluated therefore for statistical comparisons these two animals were not included, resulting in a prevalence for comparison of 5.4%. At endline there was a significant difference in prevalence in the two areas using the logistic regression model (P = 0.041).
There was a statistically significant difference between the prevalence at baseline (17.2%) and at the end of the study (0%) in the intervention area (P = 0.001) using the two-sample binomial test but non-significant difference between the baseline prevalence in the control area (15.1%) and endline (5.4%) (P = 0.092).

Tissue distribution of cysts
The distribution of viable and non-viable cysts in the various tissues examined is shown in Table 5. No T. solium cysts were detected in the liver.

Discussion
To our knowledge, this was the first evaluation of porcine cysticercosis levels in Ugandan pigs using a detailed postmortem assessment. At baseline, 16% (18 out of 111) of slaughter-age pigs from the Bukedea and Kumi districts of Eastern Uganda were found to harbour viable infections with T. solium. A similarly high rate of infection was detected by Kisakye and Masaba [6] in pigs from the Lira district of central Uganda by carcase inspections which included incisions in various muscle areas.  In animals coming from the southern Ugandan districts around Kampala, Kisakye and Masaba, the same authors [6] found no animals to be infected among 214 carcases that were examined. They suggested that the differences in prevalence were associated with animals free ranging in the Lira district but being either tethered or in pens in the districts around Kampala. In the districts where our trial was undertaken, approximately 60% of households allowed their pigs to roam freely, consistent with them potentially having access to human faeces and leading to the relatively high prevalence of porcine cysticercosis that we found. Several studies have used serological methods to assess porcine cysticercosis levels in Uganda [4,7,8], however these methods have been found to be unreliable due to cross-reactivity in the assays in pigs infected with T. hydatigena as well as high levels of positive reactions in pigs that have neither T. hydatigena nor T. solium infection [22][23][24]. Two assessments were made of animals in the intervention area after the introduction of vaccination and oxfendazole treatment, an interim assessment approximately 12 months after the 3-monthly interventions were implemented in the communities, and at the end of the trial 19 months after the interventions started. At both time points, no treated animal was found to harbour any viable T. solium cysts. This represented a statistically significant reduction in the prevalence of porcine cysticercosis in slaughter-age pigs, both in comparison to the prevalence in the intervention area at the start of the trial (P = 0.001), and in comparison to the prevalence of infection in the control area at the end of the trial (P = 0.041). There was no significant difference between baseline and end of study prevalence in the control area (P = 0.092).

Table 4 Percentage prevalence of porcine cysticercosis in control and intervention areas
Data have been published from field evaluations of TSOL18 vaccination of pigs together with oxfendazole treatment that were undertaken in Cameroon [14], Peru [17] and Nepal [15], Tanzania [16] and Zambia [18]. In each of these trials, T. solium transmission was eliminated by the pigs involved in the studies. The trials undertaken in Peru and Zambia also involved treatment of the human population with taeniacide. A further field trial tested a combination of the TSOL16 and TSOL18 antigens, achieved 99.9% protection [25]. The findings from our study undertaken in Uganda are consistent with the results from all previous trials in that combined use of the TSOL18 vaccine and oxfendazole eliminated the potential for T. solium transmission by the treated animals.
In the TSOL18 vaccine trials undertaken in Uganda and Nepal, treated pigs received both vaccination and oxfendazole treatment every 3 months. Animals sent for slaughter at about 1 year of age would have been expected to have received four treatments (e.g. at 2, 5, 8 and 11 months of age), with animals slaughtered at an older age receiving more treatments. A high frequency of treatments is required in order to ensure that new animals born into the population receive at least two vaccinations, together with at least one treatment with oxfendazole, by the time they are slaughtered for consumption.
A logical model of T. solium susceptibility in a pig population was developed by Lightowlers and Donadeu [13], with which vaccination and oxfendazole treatments were compared at different intervals for their effectiveness in prevention infected pigs transmitting the parasite. According to the model, 3-monthly treatments had the potential to prevent animals older than seven months from being able to transmit T. solium. Data accumulating from the field trials now completed in Uganda and Nepal, which adopted a 3-monthly treatment schedule, confirm the efficacy of this program of treatment to eliminate T. solium transmission by pigs.
While 3-monthly vaccination and medication of pigs may be effective in preventing porcine cysticercosis, a program involving the entire pig population may not be required in order to prevent porcine cysticercosis. Only two immunizations and a single treatment with oxfendazole are needed to provide protection to young animals [12]. In the trial conducted in Cameroon [14], vaccinated animals received three immunizations over a 12-13 month period. A pair of animals (one treated and one control) received TSOL18 vaccinations at the age of approximately 2-4 months of age (two injections four weeks apart) and a third injection approximately three months after the second injection protection assessed by necropsy 6-7 months after the third injection. Only a single oxfendazole treatment was given, at the time the animals received their second vaccination. While this was effective in eliminating the potential for T. solium infection in the treated animals, it involved a cohort of animals. It was not a program that could be applied to the whole pig population so as to ensure ongoing treatments of new-born pigs, unless a mechanism was available to ensure vaccination and medication of young pigs in the communities on a continuing basis. Practical difficulties of implementing a control program in pigs in poor rural locations so as to ensure that that all young pigs received the required treatments, led us and others to deliver the treatments repeatedly to the whole pig population. While this approach has been successful in elimination infection in the pig population this needs to be fully costed and assessed against the benefits in reduction in the incidence human T. solium infection. The feasibility of implementing vaccination and medication of young pigs, only, in Uganda and other endemic countries should also be explored and evaluated alongside other control measures in order to bring about a sustained reduction in T. solium transmission leading to a reduction in the incidence of neurocysticercosis.

Conclusions
Combined use of the TSOL18 vaccine and oxfendazole in pigs eliminated the potential for T. solium transmission in the animals involved in the trial. Future consideration needs to be given to a strategy that involves vaccination and treatments being given only to young animals in order to sustain an on-going T. solium control program.