2.1.1 Inclusion criteria.

Articles that met the following criteria were included: studies concerning Taenia spp. causing cysticercosis, animal model studies, experimental studies, studies conducted between 1^st January 1980 and 11^th October 2021. This followed the PICOS (population, intervention, comparison, outcome, and study design). (1) population- all Taenia spp. causing cysticercosis in experimental models; (2) Intervention- all infection methods in experimental models using Taenia spp.; (3) Comparison- the animals not infected with either eggs, proglottids or oncospheres; (4) Outcome- infection status of the animals, either cysticercosis positive or negative; (5) study design- experimental study designs involving Taenia spp. causing cysticercosis in animals.

2.1.2 Exclusion criteria.

Publications were excluded if at least one of the following criteria was met: (1) Studies did not concern Taenia spp. causing cysticercosis; (2) Studies were not animal models; (3) Studies were not experimental; (4) Studies conducted before 1^st January 1980 or after 11^th October 2021; (5) Studies with results outside the scope of the study questions (including general reviews on the topic).

3.4.1 Animal models used to study cysticercosis.

3.4.1.1 Taenia solium. A higher infection rate was seen in the immunosuppressed institute of cancer research (ICR) female mice (80%) as opposed to immuno-suppressed ICR males with an infection rate of 50%. Furthermore, male mice had a lower cysticerci recovery rate (0.05%) while females showed a 0.26% cysticerci recovery rate when infected with T. solium [2]. Higher infection rates were observed for T. solium (57–75%) in immunosuppressed male ICR mice following intravenous injection of oncospheres. In some studies, infection rate, cyst burden, and antibody-antigen levels were shown to increase with an increase in the dose of oncospheres inoculated in the animal [16, 23]. However, no correlation was found between the antibody concentration and the number of cysticerci recovered [33].

Additionally, lower doses of oncospheres used to achieve infection lead to a higher infection efficiency of 5.6% as opposed to 3.6% when a higher oncosphere dose of T. solium was surgically implanted in the subarachnoid space of piglets [23]. However, infection dose did not affect the development of cysticerci in the brain of rats after inoculation with T. solium activated oncospheres [24], though an increase in antigen titres due to an increase in the number of cysts was detected [28]. In contrast, the recovery rate of cysticerci decreased with an increase in the dose of T. solium eggs used [35]. Moreover, [37] demonstrated that pigs that ingested a lower dose of T. solium eggs had a 10% development of metacestodes as opposed to 0.75% in pigs that received a higher dose, however, the higher the dose, the more the larvae remained vesicular and infective for a longer duration.

The Non-immunosuppressed ICR, Balb/c, and C3H mice were not susceptible to oncospheres of T. solium. Nevertheless, following immunosuppression, the Balb/c, C3H, and C57 mice were susceptible to the oncospheres of T. solium with infection rates of 50%, 60%, and 100%, respectively, and the cysticerci recovery rates of 0%, 0.43%, and 0.12% respectively [2]. Besides, the normal C57BL/6N mice were also found to have a high infection rate of 80% with a cysticercus recovery rate of 0.02 to 2.4% [2].

Studies for T. solium using single breed of pigs namely Landrace, Landrace crossed with Yorkshire (LY) and Landrace crossed with Duroc and Hampshire (LDH) showed cysticerci recovery rates of 0.2 to 81.93%, 0 to 5.4%, and 0 to 0.71% respectively (Table 1). Infection with T. solium in 10-day old rats showed an infection rate of 83%, while 18-day old rats had an infection rate of 66%, whereas the 26-day old rats had the lowest infection rate of 25%. This study showed that the number of cysticerci detected reduced with rat age [24]. In yet another study, older pigs demonstrated some degree of resistance to infection [13].

On the other hand, [28] demonstrated that the number of viable cysts reduced with an increase in pig age (i.e. 5 months old pigs compared with 1 and 3 months old pigs). In this study, detected antigens were high in pigs infected at 1 month, thus demonstrating higher susceptibility to infection in younger pigs.

3.4.1.2 Taenia saginata. [1], found older cattle to be more resistant to T. saginata egg infection showing a lesser number of cysticerci and a higher number of calcified cysts. The cysticerci recovery rate was not reported in one of the studies [58]. As expected, an increase in the number of eggs used to infect the experimental animals increased the cysticerci recovery rate [57, 58]. [51] found an increase in serum globulins and a marked decrease in the albumin globulin ratio in the mice infected with T. saginata oncospheres. They further suggested that female BALB/c mice can be used as experimental animals for studying the host immune response in vaccine development trials. [50] exposed pigs to T. saginata eggs experimentally and no cysticerci were recovered from the experimental pigs, this resulted in negative serological tests for T. solium. In addition, [55], demonstrated a higher total number of cysts in calves infected with a single dose of T. saginata eggs compared to calves that were trickle infected by weekly oral administration of eggs for 12 weeks. Interestingly, higher cysticerci numbers were recovered from cattle infected with T. saginata eggs in regions that are not officially examined as they are considered non-preferential sites for cysticercus bovis [52]. Similarly, [53] found cysticerci in the non-traditional sites following oral inoculation of cattle with T. saginata eggs. [54] Oryan et al. (1998), found that the age of the animals and dose of T. saginata eggs influenced clinical signs and pathological changes in the calves infected with T. saginata eggs.

3.4.1.3 Taenia saginata asiatica. Susceptibility and cyst recovery of T. s. asiatica oncospheres in immunosuppressed male ICR mice following venous injection was assessed and lower infection rates were observed (14–20%). In the study for T. s. asiatica, C3H/HeN mice had the highest cysticerci recovery rate compared to the BALB/CAnN and C57BL/6N mice [27]. In the study conducted by [49] Fan et al. (1989) for T. s. asiatica, the Landrace crossed with the small ear miniature pigs (L-SEM) had the highest cysticerci recovery rate of 5.6% as opposed to 1.7% for the small ear miniature (SEM) and 0.03% for the Duroc-Yorkshire-Landrace cross pigs (DYL). In Contrast to the later study by [48] a cysticerci recovery rate of 27.1% was found for the SEM, 1.7% L-SEM, and 0.27% DYL for T. s. asiatica. However in a study for T. s. asiatica, a high infection rate was seen in the SEM (80%) with a cysticerci recovery rate of 36%, and no infection was seen in the L-SEM [56] (Fan et al., 1992b). Moreover, the SEM pigs were found as a favourable host for T. s. asiatica with a cysticerci recovery rate of 0.005 to 22% and an infection rate of 75 to 100% as opposed to the L-SEM pigs that had a cysticerci recovery rate of 1.1 to 5.6% and an infection rate of 83 to 100%. In addition, the DYL pigs had an infection rate of 100% and cysticerci recovery rate of 0.06 to 0.3%. [42], infected pigs with T. s. asiatica and found the cysticerci recovery rate of 1.4 to 22.9% in the SEM whereas the L-SEM had a cysticerci recovery rate of 16.1%.

3.4.1.4 Taenia asiatica. In addition, female severe combined immunodeficiency (SCID) mice developed cysticerci in the peritoneal cavity or under the skin after infection with oncospheres of T. asiatica whereas males did not [60]. In the study conducted by [59], cysticerci were recovered from SC inoculation of gerbils with oncospheres. However, no cysticerci were recovered from gerbils orally inoculated with eggs. In this study, the infectivity of the cysticerci was evaluated were a total of seven adult worms were recovered from the two human volunteers who ingested five cysticerci after 120 days post infection.

3.4.1.5 Taenia crassiceps. Two out of twelve mice became infected after oral inoculation with 100 eggs whereas no cysticerci developed in mice following inoculation with 500 or 5000 eggs of T. crassiceps [5]. In this study breed difference was not reported, voles had an infection rate of 50%, gerbils 34.6%, mice 17% after inoculation with T. crassiceps eggs [5].

3.4.1.6 Models inducing neurocysticercosis. Rats developed NCC following IC and OR inoculation with activated oncospheres and postoncospheres of T. solium [17, 21]. Interestingly, the dose of T. solium activated oncospheres affected the infection efficiency, where an increase in infection efficiency of 5.4% was seen in pigs that received a lower dose as opposed to an infection efficiency of 3.6% in pigs that received a higher oncosphere dose after surgical implantation of oncospheres in the cerebral subarachnoid space of the piglets [23]. Nonetheless, vesicular cysts were found in the brains of pigs following oral infection with T. solium eggs [13, 40]. In addition, cyst burden was high in the brains of pigs inoculated with a high dose of activated oncospheres in the common carotid artery as opposed to the pigs inoculated with a lower dose of T. solium [16]. In another study, rats were inoculated intracranially (extraparenchymally and intraparenchymal) with T. solium activated oncospheres to induce NCC. In this study, the route of infection and infection dose did not affect the proportion of rats that developed cysticerci in the brain [24]. Following IC inoculation of rats with T. solium oncospheres, an increased expression of genes associated with proinflammatory response and fibrosis related proteins was observed in the brain tissue of infected rats four months after infection [20].

3.4.2 Infecting material and route of infection used in the animals’ experimental models.

Various routes of infection were used in the models with some studies using a combination of the various routes and infecting material (Fig 3). The routes used to induce infection in these studies included the oral, intravenous, subcutaneous, intramuscular, intraperitoneal, intraarterial, intracranial, surgical, and intraduodenal routes. The infecting material used included eggs, proglottids, and oncospheres. Briefly, the T. solium, T. saginata, T. s. asiatica and T. asiatica eggs were obtained from gravid proglottids collected from individuals habouring the adult worms after treatment followed by a purge. Gravid segments were repeatedly washed, centrifuged, or triturated in a pestle and mortar to obtain the eggs. The collected eggs were exposed for 10 minutes to 0.75% sodium hypochlorite at 4 degrees celsius for oncosphere hatching in the studies. Additionally, gerbils and mice were SC, IP, and OR inoculated with oncospheres and later euthanased to obtain the cysticerci of T. s. asiatica [34].

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Fig 3. Chart showing route (s) of infection versus material (s) used to infect experimental animals in individual studies.

OR = oral; IV = intravenous; SC = subcutaneous; IM = intramuscular; IP = intraperitoneal; IA = intraarterial; IC = intracranial; ID = intraduodenal; S = surgical.

https://doi.org/10.1371/journal.pone.0271232.g003

Our review has shown that that the oral route was the most frequent route used in 29 out of 48 studies (60.4%) using eggs alone at various doses as the infecting material. The use of proglottids alone, both proglottids and eggs, both oncospheres and postoncospheres were reported in one study in each case (i.e. each representing 2.1% of the total included studies). The use of both eggs and oncospheres was reported in two studies, representing 4.2% of the included studies. Oncospheres were used as infecting material in 15 studies representing 31.3% of the included studies. Nine studies used a combination of routes to achieve infection in the various animals studied (Fig 3).

No cysticerci were detected via oral infection method using eggs or oncospheres but a high infection rate was achieved in mice via subcutaneous route using oncospheres in both T. s. asiatica and T. solium [41]. Also, [60] suggested that the intraperitoneal inoculation of mice with oncospheres yields a higher cysticerci recovery rate than the subcutaneous inoculation. However, infection was achieved in gerbils subcutaneously injected with hatched oncospheres whereas no infection was established when oncospheres were orally fed to the gerbils [59]. Similarly, oncospheres of T. s. asiatica were inoculated subcutaneously in SCID mice and yielded a higher cysticerci recovery rate of 0.1 to 1.1% while intraperitoneal inoculation yielded a lower cysticerci recovery rate of 0.3% [34]. Furthermore, some mice inoculated with oncospheres of T. solium via the intravenous route developed cysticerci, whereas no cysticerci developed in mice inoculated with oncospheres through the intramuscular route [36]. Gerbils and voles were observed to have higher infection rates than ICR mice after oral inoculation with eggs of T. crassiceps [5]. In contrast, the route of infection did not affect the development of cysticerci in the rats after inoculation with T. solium oncospheres [24]. The dose of infecting material used in the various studies are shown in Tables 4–7 while breed, Taenia spp. and age of animals used in the experimental models is shown in Table 8.

3.4.2.1 Description of the infecting material, dose and route used in the studies for T. asiatica and T. crassiceps. In the study conducted by [59], T. asiatica eggs and oncospheres were inoculated in experimental gerbils using the subcutaneous and oral route. However, in this study, the doses of eggs and oncospheres were not reported. Additionally, [60] infected the experimental mice with 50,000 oncospheres of T. asiatica using the intraperitoneal and subcutaneous route. [5] infected the experimental mice, gerbils and voles using 100–5000 eggs of T. crassiceps using the oral route.

3.4.3 The strengths and weaknesses of the animal models.

3.4.3.1 Strengths. Pig models have shown that experimentally infected pigs serve as the good model to study cysticercosis as the disease in pigs mimicks the human form of the disease. Pig models using whole proglottids have an advantage of mimicking the natural method in which pigs get infected. Moreover the infection date and dosage is known as opposed to natural infection. The use of monkeys to study cysticercosis has an advantage as the disease in monkeys may be useful to study the human disease due to immense similarities of monkeys with humans. Rodents such as mice and rats were used in severel models, these animals have an advantage of easier of handling and the entire sequence of their genome is known which may aid in making sound interpretations of experimental results.

Some of these experiments have demonstrated that the cysticerci established can be used to infect natural and other alternative definitive hosts for the establishment of adult worms [5, 34, 59] and can thus be used for future experiments especially in T. solium experimental studies where the collection of the tapeworm from taeniasis positive humans is quite challenging. Additionally, these studies have shown some differences in the establishment of cysticerci and resistance to infection due to sex, age, breed, and route of inoculation of the infecting material.

3.4.3.2 Weaknesses. Large animals such as cattle, pigs and goats are difficut to handle in intensive care units because they require trained personel to handle and thus make the entire experimental process laborious which may lead to several experimental errors. Laboratory mice and rats are not susceptible to natural T. solium infection making their models not good to study human cysticercosis and NCC. Moreover, some models used immunosuppressed animals to achieve experimental infection, this may present some limitations to understanding the host-parasite interaction under the normal physiological status of the host. Additionally, several experimental animals were inoculated using different routes which are not their natural routes of infection, athough infection was achieved in these models, results may not be extrapolated to understanding the normal pattern of how the hosts gets infected.

In most (if not all) studies there was insufficient reporting and non-usage of methods to reduce bias, such as sample size calculation. None of the studies reported how the sample size was calculated. Therefore, the power of the experimental studies (i.e. probability to detect treatment effect if it existed) could not be established, which may compromise the process of detecting the difference between experimental groups. Nonetheless, studies with low power were included in this systematic review because we wanted to give a full overview of animal models that have been used to study cysticercosis. In addition, it was not indicated clearly in the studies whether animals were selected at random for outcome assessment and whether outcome assessors were blinded from knowing the intervention that each animal received and thereby introducing detection bias. Furthermore, improvements are needed throughout experiments from random housing allocation and sequence generation. Moreover, the trial caregivers knew the intervention that each animal received and could thus introduce performance bias. Even though breed, sex, and age differences were reported in some of the studies, these results were not analysed statistically to determine if any significant difference existed among these risk factors of cysticercosis and thus the conclusions made may be questionable.