Effect of oxfendazole to control Taenia solium cysticercosis in pigs in Punjab state of India

Abstract

Taenia solium cysticercosis is a neglected zoonosis prevalent in developing countries including India. The disease has a substantial health impact in India. The control of this disease in pigs would benefit both veterinary and public health. In view of this, the current study was designed to know the effectiveness of oxfendazole against T. solium in pigs reared in Punjab, India. A randomized controlled trail was designed with two groups of treatment (T1 and T2) and a control group. Pigs from ten different litters were selected. Pigs (4 months of age) from the same litter were randomly assigned into these three groups. Treatment 1 (T1) group pigs received oxfendazole@30 mg/kg body weight at 4 months of age, treatment 2 (T2) received oxfendazole@30 mg/kg body weight treatment at 9 months of age and the control group (C) received placebo at 4 months of age. Blood samples were collected at three intervals at the age of 4, 9 and 12 months. Sandwich antigenic ELISA was performed to determine the serological status of disease in pigs. Three piglets in treatment 1 were found positive at 4 months but were recorded seronegative in subsequent samplings. One pig in the control group was positive at 9 and 12 months of age. Results showed promising effects of oxfendazole for the control of T. solium cysticercosis in pigs of Punjab, India.

Introduction

Taenia solium cysticercosis is a neglected zoonosis that affects pigs and human beings. Pigs serve as the intermediate host whereas humans are the only definitive host for this parasite. The cysticercus larva develops in the pig muscles and ingestion of cysticercus larva present in the infected pork causes taeniosis in man. In addition, accidental consumption of T. solium eggs by humans could lead to cysticercosis in which different organs might be affected, for example neurocysticercosis (NCC) when brain and occular cysticercosis when eye is infested (Chowdhury et al. 2014). Contaminated uncooked vegetables could also serve as a source of human cysticercosis (Mahajan 1982; O’Neal et al. 2014).

Neurocysticercosis attributes about 30% of seizure disorders in endemic areas around the world. In rural endemic villages, 10–20% of residents are found to have brain lesions consistent with NCC (O’Neal et al. 2014; Thomas et al. 2017). Human neurocysticercosis is a serious concern in India and the disease is one of the major causes for adult-onset epilepsy. Prevalence of NCC has been reported to be 4.5% in children presenting with acute onset seizure/focal neurological deficit (Kumar et al. 2017). The prevalence of epilepsy without NCC, epilepsy with NCC and epilepsy with NCC along with other causes has been reported to be 6.53/1000 persons, 3.48/1000 persons and 10.0/1000 per-sons, respectively in north India (Goel et al. 2011). NCC-associated active epilepsy have been reported to cause an annual median loss of Rs. 12.03 billion with 9.78 billion and 2.22 billion from north and south India, respectively (Singh et al. 2016).

In developing countries, several factors such as open defecation, presence of free roaming/scavenging pigs and sale of uninspected pork perpetuate transmission cycle of this parasite (Gonzalez et al. 2001). In addition, the use of massive chemotherapy, immunotherapy and health education has been limited (Gonzalez et al. 2001).

Chemotherapy of infected pigs has been proposed as an effective strategy to interrupt the transmission of T. solium cysticerci, thereby reducing the adult tapeworm infection in humans (Gonzalez et al. 2001; Prasad et al. 2008; Sarti and Rajshekar2003). The World Health Organisation and World Organisation for Animal Health (OIE) have recommended oxfendazole as the drug of choice for control of cysticercosis in pigs (WHO 2005). It has been reported that a single dose of 30 mg/kg of oxfendazole is nearly 100% effective in killing pig cysticerci within 8–12 weeks of post-treatment (Gonzalez et al. 2001; Sánchez-Torres et al. 2019). In Peru, the parasite transmission was successfully disrupted using mass chemotherapy with niclosamide in humans and with oxfendazole in pigs, along with pig vaccination (Garcia et al. 2016). A randomised placebo-controlled trial to understand the safety, tolerability and pharmacokinetics of oxfendazole has also been conducted for transition of this drug from veterinary to human use (An et al. 2019). However, similar experimental studies to understand the efficacy of oxfendazole in naturally infected pigs have not been conducted in India.

In view of this, the current study was performed to assess the efficacy of oxfendazole anthelmintic to control the incidence of T. solium cysticercosis in scavenging pigs in Punjab state of India. This study was conducted to know whether a relation exists between the intervention and the outcome.

Methods

Ethics approval

This study was ethically approved by Committee for the Purpose of Control and Supervision of Experiments on Animals, Animal Welfare Division, Ministry of Environment, Forest and Climate Change, Government of India through Institutional Ethics Committee, Guru Angad Dev Veterinary & Animal Sciences University, Ludhiana (70th meeting F. No. 25/4/2019—CPCSEA dated 20-03-2017).

Study area and population

The current study was performed in a slum area located in a peri-urban area Haibowal, Ludhiana (co-ordinates: Longitude 75.8156, Latitude 30.9233). Pig rearing is common in this area and pigs are allowed to roam and scavenge to meet their dietary requirements. There is a nullah that flows through this area and it is likely to be contaminated with human excreta. In addition, all the inhabitants residing in that area do not have access to toilets and pigs consuming human excreta is not uncommon. The study population consisted of 4-month-old pigs raised in this area that are allowed to roam/scavenge at least some parts of the day.

Sample size estimation

Assuming a prevalence of 10.41% in stray pigs (Chawhan et al. 2015), and a 5 times reduction in prevalence to 2.0%, a sample size of 20 pigs per group was estimated, using 95% level of confidence and a power of 0.29 (https://www.stat.ubc.ca/~ rollin/stats/ssize/b2.html). The experiment was designed at a lower power as most of the scavenging pig owners were illiterate and their response rate to participate (for a period of 8 months) in this study was very low. Note that a sample size of 101 pigs per group was actually required at a power of 0.80 (https://www.stat.ubc.ca/~rollin/stats/ssize/b2.html).

Experimental study design

We conducted a three-armed randomized block control trail starting with zero day and follow-up assessments carried out for piglets at the age of 4 months, 9 months and 12 months in all the three groups—treatment 1 (T1), treatment 2 (T2) and control (C) group (Fig. 1). In brief, we used pig litters/farms as blocks to control for others external factors and pigs were randomly assigned into different treatment(s) and control groups in different blocks. Overall, 60 pigs were selected belonging to 10 pig litters/farms. We assigned 20 piglets each into three groups. These pigs were monitored for a period of 8 months. The inclusion criteria included 4-month-old pigs reared in the study area and allowed to scavenge for some part of the day. The exclusion criteria included pigs dewormed with any anthelmintic before the start of this study. Pigs belonging to a particular farm/litter were allocated in different groups randomly using a random number generator.

Fig. 1.

Flow diagram of the study design used in the current study

The Group 1 piglets (n = 20) were treated with oxfendazole @ 30 mg/kg body weight at the age of 4 months(T1), Group 2 piglets were treated at the age of 9 months (T2) whereas the third group (C) received placebo (empty capsules filled with glucose).

Epidemiologic information related to number of animals in the herd, duration pigs are allowed to scavenge, sex and age of the animal and group to which animal was classified (T1, T2 or C) were recorded. Farmer experience (years) in pig rearing was also recorded.

Blood collection

For identification of piglets, ear tags were applied to all the pigs involved in this study. Blood collection was done in all the three groups in three sampling rounds at 4, 9 and 12 months of age. Blood was collected from cephalic vein into a vacutainer tubes and allowed to clot at room temperature. Serum was extracted and stored at − 20 °c until use.

Sandwich antigen ELISA

The serum samples were tested using commercially available Cysticercosis Antigen ELISA kit (Bio-X Diagnostic’s, Rochefort, Belgium) to detect circulating antigens of Taenia solium cystcerci. The results were interpreted as per the kit manufacturer’s instructions.

Descriptive analysis

Data was compiled in an excel spreadsheet (Microsoft office Excel 2010). Continuous variables were assessed for normality in the data. The variables such as farmer experience in pig rearing, herd size, and total hours pigs can roam/scavenge in a day (24 h) were not normally distributed and were transformed into categorical variables. Descriptive analysis was performed for all variables (Khatkar and Dhand 2014).

Conditional logistic regression

The conditional logistic regression was performed, facilitated by R statistical program (R statistical package version 3.4.0, R Development Core Team (2015). The association between each explanatory variable with the outcome variables of all the three groups were assessed in conditional logistic regression. The association was considered significant if the p value is < 0.05 (based on likelihood ratio test). The outcome variable was test status (seropositive or negative) of a pig during the third sampling (at 12 months of age) tested using antigenic sandwich ELISA. Explanatory variables included number of animals in the herd, duration pigs are allowed to scavenge, sex and age of the animal, farming experience (in years) and the group to which animal was classified (T1, T2 or C). The matching was indicated by the strata variable farm/herd ID.

Results and discussion

A total of 60 pigs, consisting of 20 (33.33%) for each group were enrolled. Sixty per cent of the pigs were male, majority (58.33%) of the pigs belong to farm with small herd size (< 13). Pigs selected were from 10 different farms, majority of the owners had pig rearing experience of < 12 years (68.33%). All pigs were sampled thrice (at the age of 4, 9 and 12 months) except 2 pigs that were lost to follow up after first sampling. Descriptive analyses of pigs involved in this study have been mentioned in Table 1.

Table 1.

Summary statistics for variables examined for pigs treated with oxfendazole followed by their T. solium seropositivity (Sandwich antigen ELISA test status) along with their controls

RBCT to see effectiveness of oxfendazole (n = 20 pairs)
Variable	Treatment 1 (T1)		Treatment 2 (T2)		Control (C) group
	N	%	N	%	N	%
Experience of farmer in pig rearing (in years)
Less than 12	12	60.00	16	80.00	17	65.00
More than 12	8	40.00	4	20.00	3	35.00
Total	20	100.00	20	100.00	20	100.00
Herd size
Less than 13	10	50.00	14	70.00	14	55.00
More than 13	10	50.00	6	30.00	6	45.00
Total	20	100.00		100.00	20	100.00
Animal let out for roam/scavenging (in hours)
Less than 2	13	65.00	18	90.00	18	80.00
More than 2	7	35.00	2	10.00	2	20.00
Total	20	100.00	20	100.00	20	100.00
Sex
Female	9	45.00	6	30.00	6	45.00
Male	11	55.00	14	70.00	14	55.00
Total	20	100.00	20	100.00	20	100.00

A total of four pig samples, three at 4 months of age and one pig during subsequent samplings were found positive using antigen ELISA. Interestingly, all the 3 pigs found positive in treatment 1 (T1) group at 4 months of age (first sampling) became seronegative at 9 months of age (during second sampling).

We record a 5.0% (3/60) prevalence of T. solium cysticercosis in pigs before being enrolled in this study. This is similar to the 3.96% prevalence of porcine cysticercosis reported in Punjab (Singh et al. 2018). It is to be noted that all the piglets were being reared in natural conditions and there was a chance of exposure to T. solium eggs, as the piglets were allowed for scavenging. However, one pig in the control group was found sero-positive at 9 months of age (during second sampling). Summary of ELISA results are presented in Table 2.

Table 2.

Seropositivity status of pigs enrolled in treatment and control groups in this study using sandwich antigen ELISA used for the detection of viable cysticerci of various Taenia species

Group	Treatment 1 (T1)			Treatment 2 (T2)			Control (C)
Sampling	1st	2nd	3rd	1st	2nd	3rd	1st	2nd	3rd
Positive	3	0	0	0	0	0	0	1	1
Negative	17	19	19	20	19	19	20	19	19
Lost to follow-up	0	1	1	0	1	1	0	0	0

For treatment and control groups, none of the pig was detected sero-positive in both the treatment groups at 12 months of age, whereas one pig was detected sero-positve in the Control group when tested at 12 months of age. This indicates effectiveness of oxfendazole in controlling T. solium cysticercosis in endemic areas. Results of conditional logistic regression have been presented in Table 3. However, no statistically significant associations were recorded between outcome variable (test status at the third outcome) and the examined risk factors. This might be due to small sample size used in the current study.

Table 3.

Conditional logistic-regression analysis of 20 oxfendazole treated/non-treated pig case–control pairs (Outcome: Pig test status (seropositive or negative) at 12 months of age; stratum variable: Farm/herd ID)

Factor	Beta	Standard error (Beta)	Odds ratio
Model 1(Treatment status)
Control	Referent	–	1.0
Group T1	− 22.4	54,800	1.869 × (10)−10
Group T2	− 22.4	54,800	1.869 × (10)−10
Likelihood ratio test = 2.36, p = 0.308
Model 2 (Sex)
Female	Referent	–	1.0
Male	20.8	41,800	1.079 × (10)9
Likelihood ratio test = 0.97, p = 0.324
Model 3 (time pig allowed to roam/scavenge in a day)
Less than 2 h	Referent	–	1.0
More than 2 h	0.594	1.13 × (10)8	1.81
Likelihood ratio test = 0, p = 1
Model 4 (Pig rearing experience)
Less than 12 years	Referent	–	1.0
More than 12 years	1.06	1.00 × (10)8	2.886
Likelihood ratio test = 0, p = 1

*95% CI: Inestimable

The study had certain limitations. Pigs could not be slaughtered at the end of study; therefore, accurate infection status could not be ascertained. We also had a lower sample size due to a low response from pig farmers.

Many other studies have also confirmed effectiveness of oxfendazole in controlling T. solium cysticercosis in pigs. For example, Pondja et al. (2012) found oxfendazole to be effective to control porcine cysticercosis. Gonzalez et al. (2001) also confirmed the safety and efficacy of a single dose of 30 mg/kg oxfendazole for the treatment of porcine cysticercosis. However, it has been stated that oxfendazole cannot be a stand-alone approach because in high endemic areas a certain number of animals may get infected after treatment and before slaughter (Pondja et al. 2012).

The current study indicates that oxfendazole could be effective in controlling T. solium infection in pigs in Punjab. However, the effectiveness should be further explored by conducting studies with a larger sample size and post-mortem inspection at the end of the study.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Informed consent

Informed consent was provided by the pig owners for the conduct of this study.

Research involving animals

This study was ethically approved by Committee for the Purpose of Control and Supervision of Experiments on Animals, Animal Welfare Division, Ministry of Environment, Forest and Climate Change, Government of India through Institutional Ethics Committee, Guru Angad Dev Veterinary & Animal Sciences University, Ludhiana (70th meeting F. No. 25/4/2019—CPCSEA dated 20-03-2017). Naturally reared pigs were involved in this study.