Experimental animal models and their use in understanding cysticercosis: A systematic review.

BACKGROUND: Cysticercosis and Neurocysticercosis (NCC) can be studied using several animal species in experimental models which contributes to the understanding of the human form of the disease. Experimental infections of Taenia spp. are vital in explaining the modes of transmission of the parasite and helps the understanding of transmission of the parasite in humans and thus may be useful in designing therapeutic and immune-prophylactic studies to combat the disease. Thus, this systematic review aims to explore the existing experimental animal models to the understanding of cysticercosis in both humans and animals and elucidate the risk factors of cysticercosis and identify the Taenia spp. used in these models.
METHODOLOGY: We systematically identified all publications from the Web of Science, Google Scholar, and Pubmed regarding experimental animal models using Taenia spp. that cause cysticercosis in both humans and animals. 58 studies were identified for eligibility. Of these, only 48 studies met the inclusion criteria from which data extraction was done and presented descriptively.
RESULTS: Pigs, cattle, gerbils, mice, rats, voles, monkeys, cats, dogs, and goats were used in which T. solium, T. saginata, T. saginata asiatica, T. crassiceps and T. asiatica were studied. The routes used to induce disease were; oral, intravenous, subcutaneous, intramuscular, intraperitoneal, intraarterial, intracranial, intraduodenal, and surgical routes using eggs, oncospheres, and proglottids. Besides, the establishment of infection using eggs and oncospheres was affected by the route used to induce infection in the experimental animals. The cysticerci recovery rate in all the experimental studies was low and the number of animals used in these experiments varied from 1 to 84. Although not analysed statistically, sex, age, and breed of animals influenced the cysticerci recovery rate. Additionally, the cysticerci recovery rate and antibody-antigen levels were shown to increase with an increase in the dose of oncospheres and eggs inoculated in the animals. Contrasting results were reported in which the cysticerci recovery rate decreased with an increase in the dose of eggs inoculated.
CONCLUSION: This review describes the various animal experiments using Taenia species that cause cysticercosis highlighting the animals used, age and their breed, the routes of infection used to induce disease and the sample size used, and the cysticerci recovery rate in these animal models.

1.0 Introduction

The larval stage of the Cestoda family Taeniidae known as the cysticercus causes an infection called cysticercosis [1]. The cysticerci are recovered from various tissues in the infected animals and the cysticerci recovery rate can be defined as the number of cysts recovered from the infected animals depending on the dose of the infecting material used [2]. Cysticercosis is a parasitic zoonosis that has been ranked on top of the Neglected Tropical Diseases (NTDs) list by the World Health Organization (WHO). The institution of appropriate control measures, however, still requires further research [3].

The genus Taenia contains many species that infect humans and domestic animals. Of these, T. solium and T. asiatica eggs can infect pigs if they ingest eggs excreted from human tapeworm carriers [6] whereas cattle serve as intermediate hosts for T. saginata [1]. In addition, small ruminants such as mice, rabbits, and other rodents serve as intermediate hosts for the larval stage of T. crassiceps which share the definitive hosts (i.e. dogs, foxes, wolves, and felids) with T. hydatigena [4, 5].

Taenia saginata, T. solium and T. asiatica share the same definitive host (humans). Eggs or gravid proglottids are passed with faeces of humans and cattle (T. saginata) or pigs (T. solium and T. asiatica) become infected by ingesting vegetation contaminated with eggs or gravid proglottids [6, 7]. The oncospheres hatch in the animal’s intestines, invade the intestinal wall and migrate to striated muscles, where they develop into cysticerci (T. solium and T. saginata) [6, 7]. However, T. asiatica larval stage attacks the visceral organs of the pig [8]. Humans become infected by ingesting raw or undercooked infected meat (T. solium and T. saginata) or infected liver (T. asiatica) [6]. About 2–4 months, a cysticercus develops into an adult tapeworm in the human intestine where they attach to the small intestine by their scolex. Accidental ingestion of T. solium eggs causes cysticercosis and neurocysticercosis in humans [9]. In contrast, it is not yet postulated whether T. asiatica causes hepatic cysticercosis in humans [8].

The life cycle of T. crassiceps starts in the intestines of wild carnivores where it reproduces [10]. The infective eggs are released in the faeces of the carnivores which are eaten by rats [11]. The life cycle repeats when rats harbouring the larval stage are eaten by another canine. Humans are rarely infected by T. crassiceps, if infection occurs, it causes ocular larva migrans which may result in blindness especially in immune-compromised individuals [11].

Cysticercosis caused by Taenia spp. affects several species including humans, cattle, goats, sheep, pigs, and dogs. However, cysticercosis in humans and pigs caused by T. solium is an important socio-economic problem in countries where poverty, poor sanitation, and hygiene prevail which usually favour transmission of the parasite. In humans, the most frequent form of the disease is neurocysticercosis (NCC) [12, 13].

The processes that occur during the infection course in cysticercosis and NCC can be studied in animal models that closely resemble the parasite life cycle [14, 15]. This is useful to the understanding of the pathophysiological processes, identification of specific biomarkers for early stages of development, the immune response, and pathological outcomes. Furthermore, animal models with high rates of viable cyst infections in skeletal muscles, brain, subcutaneous tissues, lungs, eyes, liver, and the heart, thyroid, and pancreas may control for variables such as infection dose [16]. Moreover, experimental models are useful to the comprehension of the host-parasite relationship and thus aid in understanding cysticercosis in both humans and animals in detail [12].

Animal models are useful for investigating the process that occur during the infection course of cysticercosis in various animal species [16]. These models help to further understand the immune response mounted by animals and may aid in the development of vaccines and help in the identification of specific biomarkers for development of disease [16]. Moreover, experimental studies of Taenia species may help in the testing of vaccines in order to interrupt the life cycle of the parasites by preventing animals from obtaining the larval stage [13].

The purpose of this study was to provide a systematic review of the existing experimental animal models (species or strain of animals used, sample sizes used, and the method of inducing disease) and the risk factors of cysticercosis. These are important because they contribute to the understanding of the human form of the disease. Additionally, the study aimed to investigate the cysticerci recovery rate in the animal models which may help future cysticercosis research experimental designs if desired results are to be obtained.

2.0 Methods

In this study, all experimental animal models using Taenia spp. that cause cysticercosis and met the inclusion criteria were included. The systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines in March 2021 [18, 19]. Information was collected on the methodology of the model (Species of animals used, infection method, infecting material used, sample sizes, sex, age, and duration of the study) model strengths and/or weaknesses, and relevant outcomes of the study. We did not register our systematic review protocol on the database PROSPERO as our data extraction process was already completed by the time we obtained knowledge on how to register the protocol on PROSPERO.

2.1 Eligibility criteria

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 1st January 1980 and 11th 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 1st January 1980 or after 11th October 2021; (5) Studies with results outside the scope of the study questions (including general reviews on the topic).

2.2 Information sources

The information was obtained from online databases; Web of Science, PubMed, and Google Scholar.

2.3 Search

The search was conducted in the three databases between 1st and 4th Dec 2020 using one search phrase, another search was conducted on 11th October 2021. The search phrase read as follows; (Experimental infection OR experimental model OR animal models OR animal experiments) AND (Cysticercosis* OR neurocysticercosis*) AND (Taenia solium OR Taenia saginata OR Taenia ovis OR Taenia hydatigena OR Taenia crassiceps OR Taenia asiatica) AND (Infection using eggs OR infection using oncospheres OR infection using proglottids).

2.4 Study selection

The PRISMA guidelines were used to select studies. Duplicates were removed from the total publications searched. The remaining publications were screened on title and abstract and those that did not meet the inclusion criteria were excluded. Full texts were then read and those that met the inclusion criteria were included for data extraction.

2.5 Data collection process

Data extraction included methodology of the model (Species of the animals used, infection method, sample sizes, infecting material, and age of the animals used) model strengths and/or weaknesses, and relevant outcomes of the study. Data extraction was done by one reviewer (MCS) while the other reviewer verified the extraction (RM). Other reviewers were consulted where there was disagreement. Data was entered in the excel spreadsheet.

2.6 Data items

The following data was extracted; Species of animals used, Taenia spp. used, age of the animals used, number of the animals used, breed or strain of the animals used, sex, author and year of publication, duration of the study, method of infection (oral, surgical or any other method employed in the study), infecting material used (eggs, proglottids and oncospheres), and the cysticerci recovery rate.

3.0 Results

3.1 Study selection

The computerized search yielded 999 articles, of which 857 were retained after removing duplicates. Based on title and abstracts, 781 titles were removed for being non-experimental studies. Data on 48 articles were included in the extraction (Fig 1 and Tables 1–8).

Fig 1

PRISMA flow diagram.

Table 1

Description of the animal models used in the included studies for T. solium and T. s. asiatica.

Author	Animals used	Sample size	Taenia spp. used	Cysticerci recovery rate/density
Carmen-Orozco et al., (2021) [20]	rats	19	T. solium	Not Reported
Palma et al., (2019) [21]	rats	36	T. solium	Not Reported
Mejia et al., (2019) [17]	rats	100	T. solium	7–32 cysts
Alroy et al., (2018) [16]	pigs	12	T. solium	0.64–1.14%
Gomez-Puerta et al., (2018) [22]	pigs	20	T. solium	2–40 cysts per pig
Fleury et al., (2015) [23]	pigs	24	T. solium	3.6–5.4%
Verastegui, et al., (2015) [24]	rats	84	T. solium	1–4 cysts per rat
Borkataki et al., (2013) [25]	pigs	5	T. solium	Not Reported
Da Silva et al., (2012) [26]	pigs	7	T. solium	3.78–81.93%
Peng et al., (2009) [27]	mice	15	T. s. asiatica	Not Reported
Deckers et al., (2008) [28]	pigs	18	T. solium	1.5–98.5%
Maravilla et al., (2008) [29]	pigs	4	T. solium	0.2–4.2%
Garrido et al., (2007) [30]	pigs	12	T. solium	Not Reported
Linghu et al., (2007) [31]	pigs	12	T. s. asiatica	Not Reported
Fan et al., (2006) [32]	pigs, cattle, goats, monkeys	83 pigs, 10 calves, 17 goats, 4 monkeys	T. s asiatica	0.005–22% pigs, 0.03–6% calves, 0.01–0.02% goats, 0.01% monkeys
Soares et al., (2006) [33]	pigs	7	T. solium	0.42–28.57%
Chang et al., (2005) [34]	mice, hamsters, gerbils	18 mice, 55 hamsters, 81 gerbils	T. s. asiatica	0.1–3% mice
Nguekam et al., (2003) [35]	pigs	14	T. solium	0.03–3.2%
Liu et al., (2002) [36]	mice	80	T. solium	1–2 cysts per mouse
Santamaria et al.,(2002) [37]	pigs	30	T. solium	0–2.5%
Verastegui et al., (2002) [38]	pigs	4	T. solium	0–138 cysts per animal
Verastegui et al., (2000) [14]	pigs	18	T. solium	0–69 cysts per animal
Wang et al., (2000) [39]	mice	20	T. s. asiatica	0.3–4.6%
De Aluja et al., (1999) [40]	pigs	13	T. solium	Not Reported
Wang et al., (1999) [2]	mice	10	T. s. asiatica, T. solium	2.4–3%
Ito et al., (1997a) [41]	mice	18	T. s asiatica, T. solium	Not Reported
Chung and Fan, (1996) [42]	pig, monkey	12, pigs, 1 monkey	T. s. asiatica	1.9–22.9% pigs, 0.8% monkey
De Aluja et al., (1996) [13]	pigs	16	T. solium	Not Reported
Fan et al., (1996) [43]	pigs	19	T. s asiatica	1–2%
Kaur et al., (1995) [44]	pigs	12	T. solium	0–600 cysts per pig
Fan et al., (1994) [45]	pigs, dogs, cats, goats, cattle	3 pigs, 4 dogs, 4 cats, 2 goats, 1 calf	T. solium	0.8% pig, 0.3% dog
Eom et al., (1992) [46]	pigs, cattle	16 pigs, 2 calves	T. s. asiatica	0–0.7% pigs, calf rate Not Reported
Fan et al., (1992a) [47]	pigs, cattle	6 pigs, 1 calf	T. s. asiatica	11% pigs, 6% calf
Fan et al., (1990) [48]	pigs	38	T. s. asiatica	0.27–27.2%
Fan et al., (1989) [49]	pigs, cattle, goats	8 pigs, 1 calf, 2 goats	T. s. asiatica	0.6–5.6% pigs, 0.03% calf, 0.02% goat

Table 8

Breed, Taenia spp. and age of animals used in experimental models.

Animals used	Breed		Age (days)	Taenia spp.
Pig	i. Duroc-Yorkshire- Landrace	vii. Yorkshire- Landrace		T. solium
				T. s asiatica
	ii. Yorkshire	viii. Landrace	5 to 730	T. saginata
	iii. Hampshire	ix. Landrace-Duroc-Hampshire
	iv. Duroc	x. Landrace- Duroc
	v. Landrace- Small Ear Miniature	xi. Landrace-Hampshire
vi. Small Ear Miniature
Cattle	Holstein	Hereford		T. solium
	Crossbreed	Angus	4 to 570	T. s asiatica
	Jersey			T. saginata
Gerbils	i. Meriones unguiculatus		35 to 98	T. asiatica
				T s. asiatica
Mice	i. Balb/CAnN	iv. ICR		T. solium
	ii. C57BL/6N	v. SCID		T. s asiatica
	iii. C3H/HeN	vi. CB17-scid	35 to 84	T. saginata
				T. crassiceps
Voles	i. Clethrionomys rufocanus bedfordiae		Not Reported	T. crassiceps
Rats	i. Holtzman			T. solium
	ii. Wistar		6 to 35	T. saginata
				T. crassiceps
Hamsters	i. Golden		21	T. s asiatica
Monkeys	i. Macaca cyclopis		360	T. s asiatica
Cats	NS		Not Reported	T. solium
Dogs	i. Mongrel		7 to 150	T. solium
Sheep	i. Soay		Not Reported	T. saginata
	ii. Texel
	iii. Four-horned
Goats	i. Saanen		5 to 13	T. solium
				T. s asiatica

3.2 Risk of bias assessment

The Systematic Review Centre for Laboratory animal Experimentation (SYRCLES’s) risk of bias tool for animal studies [61] was used to assess the risk of bias for all studies included in the review after the full-text screening (Fig 2). Two independent reviewers assessed the risk of bias in the included studies. Disagreements between reviewers were resolved through consensus. The assessment of the risk of bias showed that there was no blinding of investigators involved in enrolling participants (animals) and they could foresee the assignment of interventions and thus introduce selection bias. Moreover, there was no randomization in the selection of subjects.

Fig 2

Risk of bias in included studies using the SYRCLEs risk of bias tool.

3.3 Quality assessment of the studies included

Quality assessment of included studies was performed independently by two reviewers, blinded to the name of the authors. The quality of included studies was assessed by the two reviewers according to the Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines Checklist [62].

3.4 Synthesis of results

Several animal experimental models have been used to study cysticercosis with varying degrees of success [16, 21, 49] using varying sample sizes and ages of experimental animals. We found that researchers have used pigs, cattle, gerbils, voles, mice, rats, hamsters, monkeys, cats, dogs, sheep, and goats as experimental animals using different Taenia spp., with some experiments using a combination of these animals in the experimental models. The Taenia spp. used in these experiments included T. solium, T. saginata. T. s. asiatica, T. asiatica and T. crassiceps. One study involving mice [41] and one involving rats [21] used a combination of two Taenia spp. in which the cysticerci recovery rate was not reported. Our findings revealed that the cysticerci recovery rate in all the studies was low. Thirty-six studies (75%) did not report the sex of the animals used. Two studies used male animals only (4.2%) while four studies used female animals only (8.3%). Six studies used a combination of male and female animals (12.5%). In most of these studies, breed or strain susceptibility to Taenia experimental infection was not assessed, even in studies that reported variations in cysticerci recovery rate due to breed, the results were not analysed statistically to determine whether statistical significance existed due to breed or strain of the animals used.

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].

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.

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.

Table 4

Description of the Taenia eggs as infecting material and the dose using the oral route in the studies for T. solium.

Author	Dose
Gomez-Puerta et al., (2018) [22]	52–312
Borkataki et al., (2013) [25]	100000
da Silva et al., (2012) [26]	200000
Maravilla et al., (2008) [29]	50000
Garrido et al., (2007) [30]	100000
Soares et al., (2006) [33]	200000
Nguekam et al., 2003 [35]	1000–100000
Santamaria et al., (2002) [37]	10–100000
De Aluja et al., (1999) [40]	100000
De Aluja et al., (1996) [13]	100000
Kaur et al., (1995) [44]	5000–20000
Fan et al., (1994) [45]	2000–10000

Table 7

Description of the infecting material, dose and route such as subcutaneous (SC); oral (OR); intraperitoneal (IP) and intravenous (IV) used in the studies for T. s asiatica.

Author	Infecting material	Route	Dose
Peng et al., (2009) [27]	oncospheres	SC	5000
Linghu et al., (2007) [31]	eggs	OR	120000
Fan et al., (2006) [32]	eggs	OR	1000–30000
Chang et al., (2005) [34]	oncospheres	SC, IP	20000–40000 SC
			18600 IP
Wang et al., (1999) [2]	oncospheres, eggs	SC, IV	500–5000
Ito et al., (1997a) [41]	oncospheres, eggs	OR	NR
Chung and Fan (1996) [42]	eggs	OR	1500–30000
Fan et al., (1996) [43]	oncospheres	IV	5000–10000
Eom et al., (1992) [46]	eggs	OR	25000–890000
Fan et al., (1992a) [47]	eggs	OR	10000
Fan et al., (1990) [48]	eggs	OR	1000–100000
Fan et al., (1989) [49]	eggs	OR	1000–380000

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.

4.0 Discussion

In total, 12 animal species were used in the experimental models involving 5 Taenia spp. In these studies, infection was established in the animals through inoculation with eggs, oncospheres, and proglottids using various routes. However, the cysticerci recovery rate was low in all the cases. Our findings revealed that the dose of eggs and oncospheres, used affected the rate of infection with few contrasting results. The variations in the establishment of cysticerci in the various experiments could be due to the genetic differences of the parasites used to infect the animals as the parasites were obtained from humans and animals with different genetic makeups [29].

In a few studies that reported sex differences in the cysticerci rates, females animals were shown to be more susceptible to infection as opposed to male animals. This suggests an influence of the sex hormones in susceptibility and resistance to infection by Taenia spp. [63, 64]. Although not statistically analysed, our findings revealed that breed, sex, and age of the animal had an influence on the cysticerci recovery rate in the infected animals. The lower infection and cysticerci recovery rates in older animals may suggest that parasites get destroyed as the animal grows by the host’s innate and adaptive immune system [13].

The route of infection is an important parameter that should be considered when establishing infection in experimental animals using eggs, oncospheres, or proglottids. The results show that higher infection rates were obtained after subcutaneous inoculation of oncospheres as opposed to any other route of infection. Moreover, the cysticerci recovery rate was high in animals after subcutaneous inoculation with oncospheres. On the other hand, a higher cysticerci recovery rate was obtained when the intraperitoneal route was used as opposed to the subcutaneous route after oncosphere inoculation. To induce NCC, oncospheres were inoculated through the IC and surgical methods, and thus any other method of inoculation may not yield the desired results. Besides, eggs were orally given to experimental animals and NCC was established in very few studies whereas oncospheres were inoculated IA and cysticerci were recovered in the brains of infected animals. However, if any other method other than oral, IA, IC, and surgical is to be used, it should be investigated whether it can induce NCC. Most investigators could not induce NCC in the large experimental animals because of the uncertainty and variability of the oral infection efficacy. Moreover, the cost of purchasing large animals and long-time maintenance of the animals as opposed to laboratory animals is another limiting factor [24]. Although rats and mice are not the natural hosts of T. solium that cause NCC in humans, they can be used as models to study the human disease following IC inoculation in which the recovered cysticerci have characteristics similar to the ones observed in humans and pigs [24]. The effect of the route of infection has been shown in other studies like that of [65] where the course of infection of Brucella melitensis was investigated following inoculation of C57BL/6 mice using three different routes of inducing disease.

The other routes of infections are less convenient and many authors preferred using the oral route which has its challenges. Eggs were orally given to the experimental pigs in most of the studies and the results varied based on the dose and age of the animals. Several studies reported a low infection rate following oral inoculation of experimental animals. These low infection rates could be attributed to the fact that eggs were kept for a long time before being used to infect experimental animals [26]. Moreover, eggs were removed from the faecal material or proglottids which may play a role in achieving infection. Further, most studies did not assess egg viability before infecting the experimental animals. Therefore, they may have infected animals with eggs of questionable viability leading to low infection rates. In addition, the techniques in many studies are not standardized leading to significant variability in the models used [14]. Another limiting factor in infecting natural hosts could be the presence of maternal antibodies and thus future studies should aim at testing all experimental animals to ensure that they are free from these antibodies if high infection rates are to be achieved [35]. In some studies, eggs of the Taenia parasites were exposed to animals in which natural infection does not occur and could be one of the reasons for the lower infection rates obtained [50].

In our opinion, the number of animals used in several studies was not adequate. Some pig, cattle, rat, monkey, cat, dog, and goat studies used 1 to 4 animals which were not sufficient enough to detect any differences due to the induction of infection. Besides, it is not feasible to divide animals into control and treatment groups with inadequate sample sizes. However, mice and rat studies had a fairly good sample size as opposed to most of the large animal studies. Limited sample sizes used in some models coupled with the fact that animals were kept in well-controlled experimental conditions which are different from field conditions especially in large animal models could be another factor causing low infection rates. In natural conditions, factors such as poor nutrition, as opposed to well-fed experimental animals, may impair the immune systems of the animals and thus make them more susceptible to infection [50]. To be able to determine whether or not a significant difference exists between means or proportions observed in comparison groups, appropriate sample size is required because of its effect on statistical power. We propose that future experimental models focus on avoiding the use of inadequate sample size and other design issues. This will help investigators to make sound conclusions as it is wasteful and inappropriate to conduct a study with inadequate power [66]

5.0 Conclusion

Overall, this systematic review shows that several animal models have been used to study cysticercosis in animals using Taenia spp. with varying degrees of success. The cysticerci recovery rate differences were attributed to breed, age, sex, and the routes of inoculation used to establish infection. The poor reporting of some methodological details in the animal experiments as revealed by this review may lead to a lack of repeatability of the models and may hinder drawing well-founded conclusions from some of the studies conducted. Therefore, future animal models should be of high methodological quality to eliminate bias if our current knowledge of cysticercosis is to be improved.

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Reviewer #1: The article entitled “Experimental animal models and their use in understanding cysticercosis: A systematic review” reported the results of a systemic review without meta-analysis on the question: What are the experimental animal models and their use in understanding cysticercosis. It was a good start that the authors mentioned NCC, which would be interesting for the relevant field if the question is to identify animal models for NCC. Unfortunately, the authors didn’t form an exclusive question, thus failing to write a scientifically sound review. The purpose of a systematic review is to draw conclusions based on the evidence to answer the well-defined and narrow question. On another note, it appears to me that the authors are often confused about different species of Taenia and the diseases that may cause in humans. Also the paper was not carefully written including the references provided. Thus, I suggest the authors reconstruct their questions to improve the review.

Here are a few examples:

Line65: “T. asiatica, T. solium and T. saginata share the same intermediate host.” This is scientifically wrong. But later in Line69, authors stated “……share the same definitive host”

Line169, “infecting material used (eggs, …. cysticerci)” cysticerci should not be used for infection of the purpose.

Line244- 248, is under the subtitle Taenia solium, but, as a matter of fact, reference#57 and 58 are about T. saginata. Thus, the authors finding is not valid.

References: #8, the year is 2020, should remove 2007; #57, missing journal name

Reviewer #2: It is an interesting article where a systematic bibliographical review is carried out, of the different animal models that have been developed to study the interaction between Taenia spp. and its intermediate host. It should be accepted with some major changes

My biggest criticism is in the section on the strengths and weaknesses of the animal models, should be highlighted as a tool to understand the pathogenesis of neurocysticercosis. As the reproducibility of the animal model, the clinical picture and pathology that develops. As for example, the use of pigs to study NCC has the advantage of being the natural host, however it is difficult to have a good reproducibility of the experimental infection of the central nervous system, while the use of rats presents a high reproducibility, but has the disadvantage of not being the natural host. The use of immunosuppressed animals has many limitations to understand the host-parasite interaction. To have animal model with characteristic of clinical symptoms and pathology similar to human , it is help to study the disease.

My minor criticism is in Table 1, the study by Alan Mejia Maza (2018) should be included, where they use the NCC rat model, in which they compare two routes of infection (intracranial and oral).

On the other hand, in Table 1, in the published article by Verastegui et al. (2015), the number of cysts obtained in rats with NCC is reported.

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Submitted filename: Review PONE-D-22-01651.pdf

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Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The article entitled “Experimental animal models and their use in understanding cysticercosis: A systematic review” reported the results of a systemic review without meta-analysis on the question: What are the experimental animal models and their use in understanding cysticercosis. It was a good start that the authors mentioned NCC, which would be interesting for the relevant field if the question is to identify animal models for NCC. Unfortunately, the authors didn’t form an exclusive question, thus failing to write a scientifically sound review. The purpose of a systematic review is to draw conclusions based on the evidence to answer the well-defined and narrow question. On another note, it appears to me that the authors are often confused about different species of Taenia and the diseases that may cause in humans. Also the paper was not carefully written including the references provided. Thus, I suggest the authors reconstruct their questions to improve the review.

Authors’ response: We note here the reviewer’s comments. As outlined by the reviewer, the main question we asked in this systematic review was: What are the experimental animal models and their use in understanding cysticercosis? Thus, we aimed at identifying animal models that have been used to understand cysticercosis (including neurocysticercosis), the infection caused by larvae of the tapeworm of the genus Taenia. Therefore, the systematic review investigated experimental animal models and their use in understanding both cysticercosis and NCC. We understand that NCC is what has made Taenia solium be of public health concern and that it would have been more specific had we only restricted the review to NCC. However, this systematic review was guided by the following research questions:

1) What animal experimental models (species of animals used and method of inducing disease) have been used to understand cysticercosis?

2) What are the strengths and weaknesses of these experimental models?

3) What sample sizes have been used?

4) Have the models contributed significantly to the understanding of cysticercosis?

A meta-analysis was not done because characteristics of study populations (age, sex, breed or strain of animals) and interventions used in the selected animal models were too dissimilar to combine and therefore opted for a narrative synthesis of the findings.

The following where the data items

Name of species of animals used, number of species of animals used, Taenia species used, age of the animals used, number of the animals used, breed or strain of the animals used, sex, method of infection (oral, surgical or any other method employed in the study), infecting material used (eggs/proglottids/oncospheres) and the cysticerci recovery rate.

Here are a few examples:

Line65: “T. asiatica, T. solium and T. saginata share the same intermediate host.” This is scientifically wrong.But later in Line69, authors stated “……share the same definitive host”

Authors’ response: The error of T. asiatica, T. solium and T. saginata sharing the same intermediate host has been corrected as observed by the reviewer (see Lines 68-70 of the revised manuscript). Revised sentence reads as:‘‘ The genus Taenia contains many species that infect humans and domestic animals. Of these, T. solium and T. asiatica eggs can infect pigs if they ingest eggs excreted from human tapeworm carriers whereas cattle serve as intermediate hosts for T. saginata ’’

Line169, “infecting material used (eggs, …. cysticerci)” cysticerci should not be used for infection of the purpose.

Authors’ response: We agree with the reviewer that the studies which used initial stage cysticerci to induce NCC be removed. The two studies were thus removed from the review as suggested and cysticerci deleted from all sections were it appeared (see table 3 in revised manuscript). Descriptive statistics was redone to reanalyse the proportions because of this deletion and the concerned references were deleted from the reference list. Therefore, the reference list was re-numbered.

Line244- 248, is under the subtitle Taenia solium, but, as a matter of fact, reference#57 and 58 are about T. saginata. Thus, the authors finding is not valid.

Authors’ response: The reference 57 and 58 were misplaced during the formatting process. The two references have been deleted from lines 244-248 and now been placed under the correct subtitle of T. saginata. (See Lines 275- 278 of the revised manuscript).

References: #8, the year is 2020, should remove 2007; #57, missing journal name

Authors’ response: Reference #8 year has been corrected to 2020 (see lines 571-573 of the revised manuscript); Reference #57 missing journal name has been included (Canadian Journal of veterinary research), see line 762-764 of the revised manuscript.

Reviewer #2: It is an interesting article where a systematic bibliographical review is carried out, of the different animal models that have been developed to study the interaction between Taenia spp. and its intermediate host. It should be accepted with some major changes

My biggest criticism is in the section on the strengths and weaknesses of the animal models, should be highlighted as a tool to understand the pathogenesis of neurocysticercosis. As the reproducibility of the animal model, the clinical picture and pathology that develops. As for example, the use of pigs to study NCC has the advantage of being the natural host, however it is difficult to have a good reproducibility of the experimental infection of the central nervous system, while the use of rats presents a high reproducibility, but has the disadvantage of not being the natural host. The use of immunosuppressed animals has many limitations to understand the host-parasite interaction. To have animal model with characteristic of clinical symptoms and pathology similar to human, it is help to study the disease.

Authors’ response: The section of weaknesses and strengths has been edited to incorporate the reviewers’ suggestions, (see lines 412-442 of the revised manuscript).

My minor criticism is in Table 1, the study by Alan Mejia Maza (2018) should be included, where they use the NCC rat model, in which they compare two routes of infection (intracranial and oral).

Authors’ response: The study by Alan M. Maza 2019 has been included in Table 1 and Table 5 and cited in the section for models inducing NCC (see line 323-326 in the revised version).The publication has also been added to the Reference list (see line number 607-609 in the revised version). Descriptive statistics was redone to reanalyse the proportions because of this addition and the reference list numbering edited (see edited reference list of the revised manuscript).

On the other hand, in Table 1, in the published article by Verastegui et al. (2015), the number of cysts obtained in rats with NCC is reported.

Authors’ response: The number of cysts obtained in the rats by Verastegui et al (2015), has been included in Table 1 (1-4 cysts per rat).

Submitted filename: Response to Reviewers.docx

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27 Jun 2022

Experimental animal models and their use in understanding cysticercosis: A systematic review

PONE-D-22-01651R1

Dear Dr. Sitali,

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Reviewers' comments:

8 Jul 2022

PONE-D-22-01651R1

Experimental animal models and their use in understanding cysticercosis: A systematic review

Dear Dr. Sitali:

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Table 2

Description of the animal models used in the included studies for T. saginata.

Author	Animals used	Sample size	Cysticerci recovery rate/density
Dorny et al., (2017) [50]	pigs, cattle	5 pigs, 1 calf	0% pigs, calf rate Not Reported
Kandil et al., (2013) [51]	mice	25	1–5 cysts per mouse
Lopes et al., (2011) [52]	cattle	25	0.01–12.55%
Scandrett et al., (2009) [53]	cattle	42	0–5.42 cysts per 100gram of tissue
Minozzo et al., (2002) [1]	cattle	5	0.01–1.43%
Oryan et al., (1998) [54]	cattle	11	0.6–14 cysts per 10gram of tissue
Bogh et al., (1996) [55]	cattle	24	0–37 cysts per animal
Fan et al., (1992b) [56]	pigs, cattle	7 pigs, 1 calf	36% pigs, 3% calf
Smith et al., (1991) [57]	cattle	15	0–52 cysts per animal
Geerts et al.,(1981) [58]	sheep, cattle	8 sheep, 2 calves	Not Reported

Table 3

Description of the animal models used in the included studies for T. asiatica and T. crassiceps.

Author	Animals used	Sample size	Taenia spp. Used	Cysticerci recovery rate/density
Chung et al., (2006) [59]	gerbils	14	T. asiatica	0.1–3.2%
Ito et al., (1997b) [60]	mice	29	T. asiatica	5–202 cysts per mouse
Miyaji et al., (1990) [5]	mice, gerbils, voles, rats, dogs	22 mice, 26 gerbils, 24 voles, 4 rats, 8 dogs	T. crassiceps	5–86 cysts per animal

Table 5

Description of the infecting material, their dose and infection route, which includes intravenous (IV); subcutaneous (SC); intramuscular (IM); intraperitoneal (IP); intraarterial (IA); intracranial (IC); intraduodenal (ID);oral (OR) and surgical (S), used in the studies for T. solium.

Author	Infecting material	Route	Dose
Carmen-Orozco et al., (2021) [20]	oncospheres	IC	120
Palma et al., (2019) [21]	oncospheres, postoncospheres	IC	10–180
Mejia et al., (2019) [17]	oncospheres	IC, OR	500 IC, 20000 OR
Alroy et al., (2018) [16]	oncospheres	IA	10000–50000
Fleury et al., (2015) [23]	oncospheres	S	500–1000
Verastegui et al., (2015) [24]	oncospheres	IC	10–40
Deckers et al., (2008) [28]	whole proglottids	OR	Not Reported
Liu et al., (2002) [36]	oncospheres	IM, IV	500
Verastegui et al., (2002) [38]	oncospheres	IM	250–2500
Verastegui et al., (2000) [14]	oncospheres	IM, IP, IV, ID	250–2500
Wang et al., (1999) [2]	oncospheres	SC, IV	500–5000

Table 6

Description of the infecting material, dose and route such as oral (O) and intraperitoneal (IP), used in the studies for T. saginata.

Author	Infecting material	Route	Dose
Dorny et al., (2017) [50]	eggs	OR	5000–30000
Kandil et al., (2013) [51]	oncospheres	IP	5000
Lopes et al., (2011) [52]	eggs	OR	20000
Scandrett et al., (2009) [53]	eggs	OR	10–10000
Minozzo et al., (2002) [1]	eggs	OR	20000
Oryan et al., (1998) [54]	eggs	OR	5000–50000
Bogh et al., (1996) [55]	eggs	OR	30000
Fan et al., (1992b) [56]	eggs	OR	1000–10000
Smith et al., (1991) [57]	eggs	OR	10–10000
Geerts et al., (1981) [58]	eggs	OR	500–10000