Knowledge, practices and seroprevalence of Taenia species in smallholder farms in Gauteng, South Africa.

Porcine cysticercosis and associated human infections are endemic in Sub-Saharan Africa, Latin America, and Asia. Poor agricultural practices, sanitary practices, and lack of knowledge increase the burden of the diseases in susceptible populations. This study investigates the seroprevalence of Taenia spp. in township pigs in Gauteng, South Africa and describes knowledge and farming practices of pig farmers regarding T. solium infections. Blood samples were collected from 126 pigs in three Gauteng township areas, and analyzed for active Taenia spp. infection using the B158/B60 Ag-ELISA. Farmer questionnaire surveys were conducted in four township areas to investigate the level of knowledge and practices associated with porcine cysticercosis and neurocysticercosis. Logistic regression models were used to assess the relationship between predictor variables and the outcome variable, knowledge of porcine cysticercosis or knowledge of neurocysticercosis. Overall, 7% of the pigs were seropositive for active Taenia spp. infection. 46% of farmers practiced a free-ranging system, while 25% practiced a semi-intensive system. Latrines were absent on all farms; however, 95% of farmers indicated that they have access to latrines at home. Most farmers had no knowledge of porcine cysticercosis (55%) or neurocysticercosis (79%), and this was not associated with any of the factors investigated. The prevalence of active Taenia spp. infection was reasonably low in this study, yet the knowledge level was also low, thus calling for further educational and training programmes to prevent Taenia spp. transmission in these communities.

Introduction

Taenia species, Taenia solium and Taenia hydatigena have been reported as a cause of porcine cysticercosis in sub-Saharan Africa, Latin America, and Asia [1-4]. While T. hydatigena is not zoonotic, T. solium is the causative agent of human cysticercosis [5]. When the metacestode larval form of the parasite develops in the brain of humans it results in neurocysticercosis, which may be associated with epilepsy, seizures and other neurological disorders [6, 7]. It is a major cause of acquired epilepsy in developing countries, commonly reported in children and older people [8-10].

Studies on T. hydatigena in pigs in Africa are limited. Nonetheless, there is evidence of the parasite circulating among the pig populations in Africa [4, 11]. Of concern is the cross reaction between T. hydatigena and T. solium in serological tests which hampers efforts to quantify the true prevalence of the two parasites in the pig population [12]. New research suggests that initial prevalences of T. solium in pig populations based on serological tests could have been overestimated [13]. Notwithstanding, porcine cysticercosis still poses a significant economic loss due to the reduced market value of infected carcasses [2, 14, 15].

Human cases of taeniasis and cysticercosis have been linked to the consumption of undercooked or raw pork infested with T. solium cysticerci [16], poor hygiene and sanitation conditions and inadequate slaughtering facilities [17, 18]. Similarly, poor sanitary and hygiene conditions, and free-range systems have been associated with increased risk of Taenia spp. in pig populations [4, 19–21]. In addition, the level of education [22, 23], knowledge on livestock management [24, 25], and poor farming practices have been associated with high prevalence of Taenia spp. in pig populations [26, 27]. In contrast, mass drug administration in humans and pigs have been reported to reduce the risk of T. solium infections when implemented as an intregrated approach [25, 26, 28].

There is evidence of Taenia spp. circulating in pigs in Gauteng province, South Africa, with 14% pigs reported to be infected in selected areas [15]. In 2016, reports of illegally slaughtered pigs for human consumption and a high number of pigs with possible T. solium infection from areas under study surfaced. Furthermore, pig farmers in these areas were reported to have had poor husbandry practices and the farms were in close proximity to human settlements. In view of this, this study estimated the seroprevalence of active Taenia spp. in domestic pigs in selected areas of Gauteng and identified factors associated with infections and transmission based on knowledge and farming practices of pig owners. We hypothesize that Taenia spp. are circulating among pigs in township areas under study and that factors associated with the burden of Taenia spp. exist in these areas.

Materials and methods

Ethical considerations

Ethical clearance was obtained from the Animal Ethics Committee at the University of Pretoria, Faculty of Veterinary Science (Reference number: V070-18) as well as the Research Ethics Committee at the Faculty of Veterinary Science (REC 014–19). Further approval was obtained from the farmers before the commencement of this study. Signed informed consent was obtained from each respondent before the questionnaires were administered.

Study area

Gauteng is a province situated in the Highveld of South Africa, occupying 1.4% of the land area of the country, and has an estimated population of 15,176,115 inhabitants [29]. Backyard farming, including pig farming, constitutes 89.5% of agricultural activities in the Gauteng Province [30]. It is estimated that 29.3% of adults in Gauteng live in poverty [31]. The province can be divided into three metropolitan cities, namely; Tshwane, Johannesburg, and Ekurhuleni [32].

The study focused on four township areas, one situated in Ekurhuleni, and three in Johannesburg. These areas were selected based on reports by veterinary extension services of poor pig farming husbandry practices and proximity between farms and human settlements. In addition, there were reports of pigs in these areas being sold and illegally slaughtered for human consumption.

Study population

This study uses mixed methods to study the outlined objectives. Purposive sampling was used to identify all 56 farms and farmers to participate in this study. All pigs within the selected farms were tested provided that they met the following criteria i) Not pregnant, ii) older than 6 months, and iii) apparently healthy, therefore, making this step a probability sample [33]. The farmers were selected on the basis of known informal pig farming system, and known risk factors for T. solium [25]. A total of 56 farmers were approached through communication with the State Veterinarians and Animal Health Technicians (AHT) from the Department of Agriculture and Rural Development in Gauteng Province. The inclusion criteria for the farmers were as follows: i) must agree to be part of the study, ii) must be 18 years or older, and iii) must be involved in pig farming as an owner or employee. All (100%) of the identified farmers in each location agreed to be interviewed.

Sample size calculation for seroprevalence

The estimated sample size of 185 used to determine the seroprevalence of Taenia spp. in pigs was calculated using the following formula n: [33], where: z = 1.96, p = expected prevalence of the disease, being 14% from a study done in Gauteng [15], and α = 0.05 as the margin of error.

Data collection

Blood collection and processing

One hundred and twenty-six blood samples were collected at three locations, in the Gauteng region. Blood samples were collected from the jugular vein of pigs into anticoagulant-free vacutainer tubes. The collected samples were stored in a polystyrene cooler box containing ice and transported to the Department of Veterinary Tropical Diseases laboratory at the Faculty of Veterinary Science, University of Pretoria. The blood was centrifuged, the serum was aliquoted into 2ml cryotubes and stored at -20°C until analysis.

Serological analysis

The serum samples were tested for circulating Taenia spp. antigens using the B158/B60 Ag-ELISA commercial kit following the manufacturer’s guidelines [34]. This assay does not differentiate between specific Taenia spp. detected in the serum. In Zambia, the in house version of the Ag-ELISA had a specificity (Sp) of 94.7% and sensitivity (Se) of 86.7% [35]. A study conducted in Zambia using the B158/B60 Ag-ELISA kit reported a specificity of 67% and sensitivity of 68% which increased to 90% in carcasses with one or more viable cysticerci and 100% for carcasses with more than 10 viable cysticerci, respectively [13]. While in the same study, the Sp of 49% and Se of 86% were reported in the identification of T. hydatigena.

A recently conducted study in Tanzania reported B158/B60 Ag-ELISA Sp and Se of 82.7% and 86.3%, respectively. However, the test T. solium positive predictive value was low (35.3%) compared to negative predictive values (98.2%) [36].

Knowledge and practices among farmers

A questionnaire was used to assess the knowledge and practices among pig farmers regarding porcine cysticercosis and neurocysticercosis. The questionnaire consisted of both closed and open-ended questions. Closed-type questions included checklists and selection type questions. While open-ended questions allowed for participants to elaborate on their opinions on the different topics in the questionnaire. The questionnaire was designed in Microsoft forms.

The questionnaire covers questions related to general knowledge and practices of farmers on Taenia spp. transmission and their attitudes towards neurocysticercosis-related illnesses. It was divided into the following themes: (i) demographic profile of respondents, (ii) knowledge on Taenia spp. infection and transmission, (iii) sanitary practices, and (iv) husbandry practices.

Prior to the data collection phase, the questionnaire was pre-tested by distribution to selected employees at the Faculty of Veterinary Sciences, officials at the Department of Agriculture and Rural Development, and a small group of individuals that were representative of the targeted population. The input obtained from the pre-testing was used to further modify the questionnaires.

Interviewers were researchers from the University of Pretoria, and they were trained on how to conduct interviews and found to be competent. The interviews were conducted in different local languages depending on the language spoken in that area or the language preferred by the farmer. The common languages spoken included IsiZulu, IsiXhosa, Sesotho, Afrikaans, and English. The average duration of the interviews was about 15 to 20 minutes.

Data management and analysis

General observation

General observations about the environment at and around the smallholder pig farming were noted: proximity to households estimated in kilometers, sewage infrastructure, waste disposal, human access, and the presence of other animals.

Descriptive statistics

Seroprevalence results were managed using Microsoft excel. The proportions of positive samples and their 95% confidence interval were calculated based on location. Information from questionnaires was captured using Microsoft excel and checked for consistency and missing values.

Proportions of categoric variables and 95% confidence intervals were calculated and tabulated using the JASP software 11.1.0 (University of Amsterdam).

Thematic analysis as described by Braun and Clarke [37] was used to analyze the responses to the open-ended questions of the farmer questionnaire. The first phase was to become familiar with the questions in which the codes are to be created. Answers from these questions were then coded and where possible linked and grouped into potential themes. In addition, each theme was reviewed to assess whether it represents the underlying question. The final definitions and names of subthemes were then structured into the four main themes namely; knowledge of respondents, pig management, control practices and sanitary practices.

Principal component analysis

Principal Component Analysis (PCA) was used as a reduction tool to identify relationship among the different themes that were used as components in this method and the questions which are identified as factor loadings. Principal component analysis using an oblique rotation (Promax) was used to reduce a large set of possibly correlated variables into a smaller set for the logistic regression model. A parallel analysis was conducted to determine the number of components to be retained from the PCA [38]. The results of the parallel analysis were also visualized using a scree plot. The factor loading cut off value was set at 0.5 accounting for 25 percent of the variance of a variable. Five principal components with eigenvalues of above 1 were identified. The components were classified as follows; knowledge of respondents, deworming and sanitary practices, pig management, monitoring and control practices, and movement restriction.

Logistic regression model

Logistic regression models were fit to the data to assess the relationship between explanatory variables; town, level of education, source of income, the reason for farming, number of years farming, the number of pigs on the farm and the outcome variables knowledge of porcine cysticercosis (Yes/No) or knowledge of neurocysticercosis (Yes/No). In the first step, simple logistic models were fit between potential predictor variables and knowledge of porcine cysticercosis or neurocysticercosis. Variables that were significantly associated with the outcome at significance level, α = 0.20 were considered for inclusion in the multivariable logistic regression model. In the multivariable logistic regression, the significance level of the predictor variables was set at α = 0.05. Odds ratio (OR) and their 95% confidence intervals (CI) were then computed for all variables in the univariable and multivariable models. The goodness-of-fitness of the model was assessed using the Hosmer-Lemeshow test statistic [39].

Results

General observations

All smallholder pig farms were in close proximity to households. However, the distance between farms and households differed by a township area: in area A, farms were on average 50m away from households, in area B, farms were 150m away from households, while in area C and area D, they were about 200m away. Smallholder farms were located downstream to the households and in some areas, sewage could be seen running along the streets and between houses towards the smallholder farms. Furthermore, there were dumping sites with used disposable diapers in sight located in close vicinity to the pig farming sites which were used for waste disposal. Children, livestock and dogs all had access to these dumping sites. The same dumping sites were used for the disposal of pig carcasses (Fig 1).

Fig 1

Dumping sites around pig farms in township areas in Ekurhuleni District, Gauteng Province, South Africa.

(A) A pig feeding around sewage; (B) Dogs and pigs roaming or feeding around dumping sites.

Seroprevalence

Although the calculated samples size for this study was 185, only 126 pigs met the inclusion criteria outlined in the methods section. Seven percent (7%, 9/126) of pigs in this study were positive for Taenia spp circulating antigens. There was no significant association between the presence of Taenia spp. infection and township area (Table 1).

Table 1

Seroprevalence of Taenia spp., as determined by the Ag-ELISA, in pigs from farms in Gauteng Province.

	Animals tested		Animals positive for Taenia species
Area	Tested	Proportion	Positive	Percentage	95% CIa
D	72	57.1	6	8.3	3.88	17.01
C	36	28.6	2	5.6	1.54	18.14
B	18	14.3	1	5.6	0.99	25.76
Total

a95% Confidence interval

Demographic profile of respondents

Almost 90% of the respondents in this study were male, and 52% had primary education or lower while 48% had secondary education or higher. Pig farming was the main source of income for the majority (95%) of respondents. Thirty percent (31%) of the respondents kept less than 10 pigs, while 48% of the respondents kept 11–20 pigs, and only 21% of respondents kept more than 20 pigs (Table 2).

Table 2

Demographic profile and farming practices of smallholder farmers in four township areas in Gauteng (n = 56).

Variable	Category	Percent
Gender	Female	11
	Male	89
Age group (years)	25–40	25
	41–60	46
	>60	29
Education	≤Primary	52
	≥Secondary	48
Source of income	Farming	94
	Employed	4
	Pensioner	2
Number of pigs kept	<10	31
	11–20	48
	>20	21
Production system	Free-range	47
	Intensive	29
	Semi-intensive	25
Source of current pig stock	Born on farm	77
	Auction	23
Introduction of new pigs	No	86
	Yes	14
Pig feed source	Kitchen waste	23
	Commercial feed	2
	Kitchen waste and commercial feed	75
Deworming of pigs	Yes	68
	No	32
Frequency of deworming	Once a year	26
	More than once a year	74
Treatment coverage	All Pigs	63
	Adult pigs	18
	Piglets	18
Name of dewormer	Don’t know name	26
	Ivermectin	66
	Tetracycline	8
Access to latrine	Yes	95
	No	6
Frequency of usage	Always	89
	Sometimes	11
Hand washing method	Water only	93
	Both water and soap	5
	None	2

Farming practice and sanitation among farmers

Forty-six percent (46%) of farmers indicated that they practiced a free-ranging system while 25% mentioned they practiced a semi-intensive system. The majority (77%) of all the farmers indicated that their animals were born on the farm. Most (86%) farmers did not introduce new stock on their herds. Of those that introduced new stock (14%), they indicated that they sourced the animals from neighboring farmers (77%) and auctions (23%). Most pigs (75%) were fed mainly on kitchen waste mixed with commercial feed (Table 2).

Respondents (68%) indicated they dewormed their pigs more than once a year (74%). Out of those that dewormed pigs, 66% used ivermectin, while 26% did not know the type of drug they used for deworming, and on the other hand, 5% used antibiotics for deworming. Most farmers (65%) indicated that they deworm all the pigs while 18% deworm only piglets or adults (Table 2). For farmers that did not deworm their pigs, the reasons given included the cost of treatment and dependency on the government for treatments.

None of the areas visited had latrines available around the farms. However, the majority (95%) of farmers indicated that they have access to latrines at home and always used them (89%). Farmers that occasionally used latrines (11%) indicated that they additionally used bushes for defecation or the open fields. Most (93%) respondents used plain water to wash their hands after using the latrine or defecating in the bushes. Only 5% of the respondents used both soap and water to wash hands after using the latrine. Two respondent did not wash hands after using the latrine (Table 2).

Knowledge of porcine cysticercosis and neurocysticercosis among farmers

Fifty-five percent (55%) of the respondents indicated that they have no knowledge of cysticercosis in pigs, and 79% had never heard about neurocysticercosis. Those who had knowledge about cysticercosis (45%) indicated that they obtained it through agricultural workshops, auctions, and media. Sixteen (16%) percent of respondents knew both how the pig acquired the disease and how to detect cysts on pigs. However, only 14% knew where to find the cyst in a pig (Table 3).

Table 3

Questions relating to knowledge porcine cysticercosis and neurocysticercosis among farmers in four township areas in Gauteng (n = 56).

Variable	Category	Number	Percent
Heard about porcine cysticercosis	Yes	25	45
	No	31	55
If Yes from where	Workshop	8	32
	Auction	12	48
	Media	5	20
Heard about neurocysticercosis	Yes	12	21
	No	44	79
Have you received training on Taenia species	Yes	12	21
	No	44	79
Do you know how pigs acquire T. solium	Yes	9	16
	No	47	84
Do you know where to find cysts in a pig	Yes	8	14
	No	48	86
Do you know how to detect cysts in a pig	Yes	9	16
	No	47	84
Farming purpose	Selling live pigs only	38	68
	Own consumption	1	2
	Selling pork meat	6	11
	All three	11	20
Slaughter pigs at home	Yes	32	57
	No	24	43
Do you perform meat inspection	Yes	17	30
	No	39	70
What do you look for when inspecting	White nodules	10	59
	Pleuritis	2	12
	Discolouration	2	12
	Cyst	2	12
	Milk spots	1	6
Action when abnormalities are found	Discard	28	88
	Sell	1	3
	Consume	3	9

The majority (68%) of the respondents indicated that they sold live pigs only and only 2% slaughtered pigs solely for their own consumption. Fifty-seven (57%) of farmers indicated that they slaughtered pigs at home for own consumption or selling yet only 30% performed meat inspection. When asked what they looked for during meat inspection, the farmers mentioned white nodules on the offal, carcass, liver, trachea, lung, and tongue. Some farmers mentioned that they looked for pleuritis, discoloration of offal and liver, cysts in the muscle and milk spots on the liver. Most (88%) respondents indicated that if an abnormality is found in the carcass, the meat is discarded by either burning the carcass and burying or feeding the carcass to the dogs. One person indicated that they sell the meat as it is and three respondents indicated they consumed the meat anyway with one saying (Table 3).

“meat is meat, it cannot be thrown away”.

In the univariate model, only the level of education of the respondents and pig farming purpose were considered for the multivariate model.

In the multivariable model, there was no significant association between level of education (p = 0.0929), pig farming purpose (p = 0.1286) and outcome, knowledge of neurocysticercosis (Table 4).

Table 4

Predictors of knowledge of neurocysticercosis.

Predictors	Univariable model				Multivariable Model
	ORa	95% CIb		p-value	ORa	95% CIb		p-value
Town&				0.6509
D	0.79	0.18	3.39	0.6631
C	0.32	0.03	3.56	0.3697
B	Ref	-	-	-
Education
None-Primary	0.38	0.10	1.45	0.1571	0.30	0.08	1.22	0.0929
Secondary-Tertiary	Ref	-	-	-	Ref	-	-	-
Source of Income
Farming	0.67	0.15	3.03	0.5996
Farming and additional jobs	Ref	-	-	-
Pig farming purpose
Selling live pigs	2.86	0.56	14.70	0.2090	3.74	0.68	20.49	0.1286
All others	Ref	-	-	-	Ref	-	-	-
Years in pig farming	1.10	0.93	1.31	0.2620
Pig numbers	1.00	0.99	1.02	0.6676

&: A was removed due to all participants responding “no” to this question

aOdds ratio

b95% Confidence interval

The results of the univariable model shows no significant association between town, level of education (p = 0.6109), source of income (p = 0.5395), pig farming purpose (p = 0.2611), years in pig farming (p = 0.4600), pig numbers kept by farmers (p = 0.8240) and outcome, knowledge of porcine cysticercosis (Table 5).

Table 5

Univariable model for predictors of knowledge of porcine cysticercosis.

Predictors	Univariable model
	ORa	95% CIb		p-value
Town				0.5381
A	0.39	0.06	2.70
D	0.93	0.25	3.52
C	1.94	0.32	11.76
B	Ref	-	-	-
Education
None or primary	0.76	0.26	2.19	0.6109
Secondary or tertiary	Ref	-	-	-
Source of Income
Farming	0.65	0.17	2.55	0.5395
Farming and additional jobs	Ref	-	-	-
Pig farming Purpose
Selling live pigs	0.52	0.17	1.62	0.2611
All others	Ref	-	-	-
Years in pig farming	1.06	0.91	1.23	0.4600
Pig numbers	1.00	0.98	1.02	0.8240

aOdds ratio

b95% Confidence interval

Discussion

The objectives of this study were to investigate the seroprevalence of Taenia species in pigs as well as knowledge and practices associated with porcine cysticercosis and neurocysticercosis among smallholder pig farmers in Gauteng.

Seroprevalence of Taenia spp.

We observed a lower (7%) proportion of pigs positive for Taenia spp. infections in this study compared to the 14%, 34%, and 54.8% reported in Gauteng, Free State [15], and Eastern Cape [40] areas, respectively.

The presence of pigs infected with Taenia spp. can be attributed to poor sanitary conditions such as sewage spillage and the presence of open disposal of waste sites accessible to the pigs observed in this study. Moreover, farmers (71%) practiced a free-roaming pig system which has been previously reported as a risk factor for the transmission of Taenia spp infections [25, 41–43]. In addition, since dogs and pigs coexist in the same environment, the pigs could be infected with either T. solium or T. hydatigena or both Taenia spp. Although the results of this study indicate the presence of Taenia spp. among pigs in these communities, further diagnosis using carcass dissection and molecular confirmation using PCR is required to confirm infection with either T. solium or T. hydatigena or both since the Ag-ELISA used is genus-characteristic [44, 45].

The proportion of infected pigs in this study did not differ between the three townships tested. Similarly, Tsotetsi et al. [15] in South Africa and Sikasunge et al [46] reported no regional significant difference in the prevalence of porcine cysticercosis. In contrast, variations in the prevalence of Taenia spp. infections have been reported among villages in Tanzania [42].

The results suggest that factors influencing the epidemiology of Taenia spp. in pigs from smallholder farms in Gauteng are similar across the study locations. In view of this, mitigation strategies such as confinement of pigs to limit access to the dumping sites and management of contaminated water sources must be applied to all the smallholder farms in the studied township areas [29].

Demographic profiles and farming practices

Th majority (75%) of the farmers in this study were above 40 years old. Myeni and others [47] reported similar findings in the Free-State province smallholder farming communities. It was surprising to see that almost half (48%) of farmers had secondary education or higher contrary to the findings of other studies which reported a lower level of education among smallholder farmers in South Africa [31, 47, 48]. The characteristic profile of the population in this study suggests that risk communication and control strategies for Taenia spp. infection and transmission must be targeted towards middle aged males. In addition, it is likely that this targeted population will be under-resourced and unable to afford expensive control programs as observed in this study.

Most farmers indicated that they deworm their pigs at least once a year using ivermectin. Similarly, farmers in Zambia and Tanzania routinely used ivermectin to deworm their pigs [24]. The proportion of farmers using unknown medicine and antibiotics as regular deworming drugs is alarming and an indication of the poor knowledge level among farmers.

Although this suggests that farmers are aware of the importance of animal health, the anti-parasitic drugs used are not effective against Taenia spp. infections. In view of this, farmers should be educated on the treatment coverage of anti-parasitic drugs with emphasis on the usage of drugs reported to be effective in Taenia spp. treatment such as, oxfendazole. Farmers should also be discouraged from buying animals from sources such as auctions, where the health status of pigs is often unknown.

Risk factors for Taenia spp. transmission

Few (6%) farmers in this study did not have access to toilets, which is comparable with the 16% reported among rural farmers in a previous study in Gauteng Province [25]. Although this is a positive outcome, access to latrines does not guarantee their use [28, 40, 42, 49]. Nonetheless, the results of this study suggest that access to latrine may not be a major risk factor for the occurrence of cysticercosis in Gauteng since most farmers had access to latrines. Notwithstanding, farmers who defecate on the open field must be made aware that this practice is likely to increase the risk of environmental contamination and the prevalence of T. solium among pigs in the area [15, 25, 28].

Informal slaughter of pigs was common (57%) among farmers in this study, and the majority of the slaughter was performed without meat inspection (70%). Similarly, a previous study in Gauteng reported that 85% of smallholder farmers did not slaughter their livestock in a registered abattoir [25]. Although the sensitivity of meat inspection for porcine cysticercosis is very low [35], the need for meat inspection as a control measure cannot be overemphasized as availability and adequacy of meat inspection services are important mitigation steps for Taenia spp. infection and neurocysticercosis [26, 50, 51]. Moreover, farmers in this study indicated that when performing inspection, they identified white nodules, pleuritis, and cysts.

In addition, the consumption of infected pork as reported by few (11%) farmers and feeding of infected meat to the dogs must be discouraged as this also may facilitate transmission of T. hydatigena [4, 52, 53]. Furthermore, children, dogs, and pigs shared the same environment in this study. Therefore, it is possible that this interface plays a significant role in the epidemiology of Taenia spp. infections in this environment [40, 54, 55]. Therefore, further studies are needed to investigate the burden of neurocysticercosis among children and consumers of pork meat sources from these areas. In addition, communities must be educated on measures that they can implement to minimize the risk of Taenia spp. infection. Farmers and consumers must be educated on the implications of selling, purchasing and consuming possibly infected uninspected meat.

Knowledge on porcine cysticercosis and neurocysticercosis

Almost half (45%) of the respondents had heard about porcine cysticercosis compared to those that were aware of neurocysticercosis which was similarly reported in Uganda [48] and Tanzania [56]. In addition, almost all farmers interviewed did not know how the pig acquires the infection. This may be attributed to a lack of education on the epidemiology of Taenia spp. observed in this study which has been previously reported as a risk factor for T. solium transmission [48]. These results are not surprising as lack of awareness on the epidemiology of T. solium has previously been reported in other African countries with 0.6% in Burkina Faso [26] and 28.6% in Cameroon [27]. Moreover, respondents in this study could not link the identified postmortem abnormalities with the potential health risks involving Taenia spp. In view of this, the use of media, workshops, and veterinary extension is encouraged as improved knowledge has been linked to reduced levels of T. solium infections [57, 58]. These knowledge transfer outreach strategies should not be targeted towards a specific group, age, gender or practice since these were not significant factors influencing the level of knowledge on porcine cysticercosis or neurocysticercosis in this study.

This study is not without limitations, the reported African Swine Fever outbreak in between April and July 2019 restricted movement between areas hence pigs from township area A could not be sampled. In addition, pig farmers were unwilling to sell their pigs therefore pig carcasses or offal could not be purchased for further analysis including carcass dissection and PCR. The Ag-ELISA used in this study was not species-specific therefore we could not differentiate infections with T. hydatigena and T. solium.

Conclusions

The Taenia spp. seroprevalence in this study was low compared to that from other studies conducted in South Africa and other African countries. Knowledge level of farmers regarding Taenia spp. in this study was low and did not differ based on education, level, and years in practice. Factors previously associated with the epidemiology of taeniasis and neurocysticercosis including free-roaming of pigs, lack of meat inspection, lack of knowledge of the disease and sewage spillage were identified in this study. Therefore, there is a need for education and training of farmers on the epidemiology of porcine and human cysticercosis to mitigate the risk of Taenia spp. infections among farmers and consumers in Gauteng township areas.

Consent form for farmers to participate in the study.

(PDF)

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(PDF)

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29 May 2020

PONE-D-20-14308

Taenia species in smallholder farms in Gauteng, South Africa: seroprevalence in pigs, and farmers’ knowledge and practices

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Comments to the Author

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Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

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5. 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: This manuscript describes a study which undertook serology on pigs living in a region of South Africa as well as a survey of farmers knowledge and attitudes related to the transmission of Taenia solium. While the manuscript’s title and abstract correctly refer to the serological outcomes as providing evidence of infection with Taenia spp, rather than T. solium specifically, all other aspects of the paper are presented as if the work was relevant specifically to T. solium. Unfortunately, the study did not obtain any direct evidence that any of the pigs involved actually had T. solium infection. The serological test that was used in the study is unable to differentiate infection with T. solium from infection with T. hydatigena. Infection with T. hydatigena has no relevance as a zoonotic infection nor would it have any relevance to the survey that was conducted. The value of the study would have been increased enormously had the Ag-ELISA positive pigs been purchased and Taenia infection, or more importantly, T. solium infection been confirmed at necropsy.

The limitations to the interpretation of the serological data in relation to T. solium place a major constraint on the importance of the study. Nevertheless, the results are worthy of publication, but the many interpretations and analyses relating to T. solium are not warranted. The valuable data about both seroprevalence and farming practices are publishable as statements of fact, without the extensive analyses that assume the data relates to T. solium when this has not been determined. Hence my recommendation is that the manuscript be re-drafted and shortened substantially.

The following are some notes to be considered if a revised manuscript is prepared.

Line 101. Add full stop.

Lines 111-114. Reference 33 does not provide the methodology that was used for the commercial Ag-ELISA. The commercially available test uses a methodology significantly different to the in-house test developed by the Institute for Tropical Medicine in Belgium which was used in the work described in Reference 33. The test’s specificity and sensitivity, referred to in Reference 11, uses the in-house test methodology and not the commercial kit. To my knowledge there has been no proper evaluation of the sensitivity and specificity of the commercial kit. There have been numerous reports of the sensitivity and specificity of the B158/B60 Ag-ELISA, several of which have found the test to have a substantially lower specificity for detection of T. solium infection than the single reference chosen to be cited here. The choice of this single reference gives a biased impression of the test’s performance.

Lines 284-6. The authors cannot attribute the prevalence of pigs infected with Taenia spp to the conditions they describe because they have no idea whether any were actually infected with T. solium. It is possible that all the positives were infected with T. hydatigena or, given the occurrence of false positive pigs in the Ag-ELISA, they may be pigs with no Taenia infection at all. The authors should qualify these interpretations by stating “If the Ag-ELISA positive pigs were infected with T. solium….” and include a statement indicating that the test positive animals may instead harbour T. hydatigena, in which case it would be an indication of the presence of dogs in the community which were not regularly treated with a taeniacide which had access to offal from pigs, sheep or goats.

Lines 288-291. It is incorrect to say that they would need carcass dissection plus PCR to differentiate animals infected with T. solium and T. hydatigena. For the vast majority of infections with either of these two parasites the identity of the species involved is clear based on the cysticercus morphology and site of encystment. It is recommended that the words ‘coupled with PCR’ be deleted.

Lines 296-298. Care needs to be exercised in commenting on the results of other publications which involved use of serology for determining the prevalence of T. solium. For may years the Ag-ELISA test was used and the fact that the test is not species specific was virtually ignored and the results assumed to relate to T. solium. Recent detailed evaluations of the test’s performance have revealed that there is a high proportion of animals that are positive in the test that have neither T. solium nor T. hydatigena infected. For example, Chembensofu et al 2017 (DOI 10.1186/s13071-017-2520-y) found that from 30 pig carcasses that were fully dissected and found negative for T. solium, the Ag-ELISA returned ten positives, of which two were infected with T. hydatigena. That is 8/30 (26.6%) animals that had neither T. solium nor T. hydatigena infection tested false positive in Ag-ELISA. With a false positive rate of this magnitude, it is not inconceivable that all 9 Ag-ELISA positive animals from the 126 that were tested in the study described in this manuscript may have no Taenia infection at all…

Lines 352-353. The sharing of the environment of children and pigs with dogs has nothing whatsoever to do with transmission of T. solium.

Lines 400-401. It is difficult to understand which authors did what – it appears that the letters refer to authors’ Christian names. It is suggested to use all the authors’ initials.

Reviewer #2: MANUSCRIPT REVIEW REPORT

Dear Editor (PLOS ONE)

Kindly find a REVIEW REPORT for a manuscript titled “Taenia spp. in smallholder farms in Gauteng, South Africa: seroprevalence in pigs, and farmers’ knowledge and practices”. The report contains general comments as well as specific comments for the different sections.

GENERAL COMMENTS

The authors of this manuscript are reporting findings on the epidemiology of an important zoonotic parasite of pigs. Somewhere, in the introduction (lines 64 – 66) and in the discussion (lines 280-283) the authors clearly indicate that similar studies have been done in the same area. In my opinion, results of an undertaking reported here are just a duplication of something which has already been reported and doesn’t add any value. It is however upon the journal Editorial Board to decide on the fate of this manuscript.

Technically, the article is fairly well written although there are some minor language problems and some study design issues which are raised in section reviews.

COMMENTS BY SECTION

TITLE

The authors need to recast the title, they could write it better than the way it is currently

ABSTRACT

Line 15-16: The sentence “Poor agricultural practices, and sanitary practices, and lack of…………” should be re-written to reduce the number of “ands”

Line 22-25: The sentence “Descriptive statistics were run and logistic regression models were used to assess the relationship between socio demographic and pig farming conditions and knowledge of porcine cysticercosis and neurocysticercosis.” should be re-written to reduce the number of “ands”

INTRODUCTION

Lines 64 – 66: the authors could not clearly indicate the rationale of conducting the study

MATERIALS AND METHODS

In line 86 the authors indicate the use of mixed method which are not evident in the subsequent part of the study design.

In line 86-87 the authors state that they used purposive sampling to identify farms and farmers to participate in this study, they should clearly explain/reason justification for this.

Line 87-90: the authors explain the selection procedure of pigs to be involved in the study and they eventually refer to the step as being probability, THIS IS SERIOUS.

Line 87-88: the authors indicate that all pigs that met criteria in all the selected farms were sampled, what was the essence of calculating the sample size then?

The authors also need to state the number of farms from which pigs were sampled;

Line 90-95: the authors explain the selection procedure of farmers. It is not clear whether these were the owners of the farms where pigs were sampled or otherwise.

Line 98: the authors indicate that the calculated sample size was 185 pigs, but in Line 101: they indicate they sampled 126 pigs, this needs explanation.

Lines 103-114: The authors do not consider “Blood collection and processing” and “Serological analysis” as part of data collection, can they give a reason to this?

Lines 128-131: The pretesting of the questionnaire doesn’t seem to make sense as the population to which it was tested is very different in characteristics from farmers. The authors need to defend their decision.

Line 150: The sentence “The open-ended questions were analyzed using thematic analysis” should be cancelled.

RESULTS

Generally this section is poorly organized, the flow is not good

In several occasions there is double presentation of results in text and in table

DISCUSSION

Generally the discussion is poorly organized

References “7” cited in line 280 and reference “36” cited in line 283 indicate that similar works were conducted in the same area. This is a weakness as resources devoted in this study could be deployed elsewhere

CONCLUSIONS

The third word in line 385 (training) does sound good to me as it is linked to imparting farmers knowledge on epidemiology of the parasite

Ethical considerations

Line 389-390: Check this “The Animal Ethics Committee Research Ethics Committee”

Line 393-394: I am doubting on the statement that; “Signed informed consent was obtained from each respondent before the questionnaires were administered”. This implies that all farmers could read and write

Acknowledgements

Line 396: The statement “The author would like to acknowledge the Gauteng……….” Indicates that this manuscript is single authored

Reviewer #3: The manuscript is interesting and very well written. I have a few comments:

All the manuscript always refers to Taenia spp, but since all the study was performed in pigs and since it also states human neurocysticercosis, I recommend to change spp for solium, mainly because it is more accurate even though the species was not identified but Taenia saginata is only found in cattle and it does not cause neurocysticercosis in humans and using T. soliun more precise in educational terms. An exception is lines 290-291

The authors should include the concept of KAP because they use it but it is not included as a specific issue such as in lines 117, 127, 157.

The reference to the statistic test cited on lines 182-183 should be provided.

Line 210 does not state pigs in numbers between 10 and 20. The authors should add: 11-20 pigs, 48%

Line 228, if not plain water, what other source/type of water did the farmers use?

Table 3. farming purpose “all three” should be moved two lines below

Line 310 eliminate “of age”

The subject of KAP should also be included in the paragraph that starts on line 359

In acknowledgments I would add to the farmers that participated in the study

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20 Aug 2020

Reviewer 1

Comments: While the manuscript’s title and abstract correctly refer to the serological outcomes as providing evidence of infection with Taenia spp, rather than T. solium specifically, all other aspects of the paper are presented as if the work was relevant specifically to T. solium. Unfortunately, the study did not obtain any direct evidence that any of the pigs involved actually had T. solium infection. The serological test that was used in the study is unable to differentiate infection with T. solium from infection with T. hydatigena.

Responses: We thank the reviewer for the comments, and we have subsequently amended the introduction section of the manuscript in line with the suggestions made. Please also see our responses below.

Comments: Infection with T. hydatigena has no relevance as a zoonotic infection nor would it have any relevance to the survey that was conducted. The value of the study would have been increased enormously had the Ag-ELISA positive pigs been purchased and Taenia infection, or more importantly, T. solium infection been confirmed at necropsy.

Response: We agree with the reviewer that purchasing of positive cases would have contributed significantly to the finding of this study. We highlighted this aspect as one of the limiting factors in the discussion section of the manuscript. We have also indicated that we could not purchase any animals due to movement restriction as results of the African Swine Fever outbreak.

Although we agree that the necropsy could have assisted in the identification T. solium, it is important to note that this method has been shown to underestimate the presence of T. solium in carcasses with very few cysts:

Gavidia, C.M., Verastegui, M.R., Garcia, H.H., Lopez-Urbina, T., Tsang, V.C., Pan, W., Gilman, R.H., Gonzalez, A.E. and Cysticercosis Working Group in Peru, 2013. Relationship between serum antibodies and Taenia solium larvae burden in pigs raised in field conditions. PLoS Negl Trop Dis, 7(5), p.e2192.

Lightowlers, M.W., Assana, E., Jayashi, C.M., Gauci, C.G. and Donadeu, M., 2015. Sensitivity of partial carcass dissection for assessment of porcine cysticercosis at necropsy. International journal for parasitology, 45(13), pp.815-818.

Comments: The many interpretations and analyses relating to T. solium are not warranted.

Response: The reviewer’s comments have been noted. However, it is important to highlight that the focus of the study was to investigate the prevalence of Taenia spp. and assess the level of knowledge of taeniasis and cysticercosis in relation to T. solium specifically.

Comments: The valuable data about both seroprevalence and farming practices are publishable as statements of fact, without the extensive analyses that assume the data relates to T. solium when this has not been determined. Hence my recommendation is that the manuscript be re-drafted and shortened substantially.

Response: We thank the reviewer for the comment. The analysis relates to factors associated with knowledge of neurocysticercosis and porcine cysticercosis. Please see the objectives of the study.

Comments: Lines 111-114. Reference 33 does not provide the methodology that was used for the commercial Ag-ELISA. The commercially available test uses a methodology significantly different to the in-house test developed by the Institute for Tropical Medicine in Belgium which was used in the work described in Reference 33. The test’s specificity and sensitivity, referred to in Reference 11, uses the in-house test methodology and not the commercial kit. To my knowledge there has been no proper evaluation of the sensitivity and specificity of the commercial kit. There have been numerous reports of the sensitivity and specificity of the B158/B60 Ag-ELISA, several of which have found the test to have a substantially lower specificity for detection of T. solium infection than the single reference chosen to be cited here. The choice of this single reference gives a biased impression of the test’s performance.

Response: This has been noted. The supporting literature for the different specificity and sensitivity values of the test have been provided. Although the commercial test has not been systemically validated, and the literature on the B158/B60 Ag-ELISA pertains to the in house test, the commercial test differs from the in house test only by a shorter procedure, a different substrate and a different method of calculating the cut off. The monoclonal antibodies in both assays are similar. The company commercialising the Ag-ELISA has done a validation of their kit against the in-house test. Updated comparative results of the B158/B60 Ag-ELISA against validated methods have been provided by Chembensofu et al., 2017. In their study, the authors show that sensitivity of the Ag-ELISA to detect carcasses with viable cysticerci (1 or more) was estimated at 91% (95% CI: 71–99%), and increased further to 100% (95% CI: 75–100%) for carcasses with at least 10 viable cysticerci. The specificity of the Ag-ELISA to detect infected carcasses was estimated at 67%.

Comments: Lines 284-6. The authors cannot attribute the prevalence of pigs infected with Taenia spp to the conditions they describe because they have no idea whether any were actually infected with T. solium. It is possible that all the positives were infected with T. hydatigena or, given the occurrence of false positive pigs in the Ag-ELISA, they may be pigs with no Taenia infection at all. The authors should qualify these interpretations by stating “If the Ag-ELISA positive pigs were infected with T. solium….” and include a statement indicating that the test positive animals may instead harbour T. hydatigena, in which case it would be an indication of the presence of dogs in the community which were not regularly treated with a taeniacide which had access to offal from pigs, sheep or goats.

Response: The comments have been noted. The sentence has been revised as “In addition, since dogs and pigs coexist in the same environment, the pigs could be infected with either T. solium or T. hydatigena or both Taenia spp.”

Taenia spp. comment, there is sufficient literature to support the assumption that the existing predisposing factors in these areas contribute to the presence of Taenia spp. in this study. Taenia hydatigena similar to T. solium have been reported in conditions where pigs are free-roaming and scavenging with increased exposure to contaminated pastures, feeds, and water. In addition, poor management practices have been reported to facilitate T. hydatigena transmission in areas where dogs or wild animals share the same environment. There are also studies that have reported co-infection between T. hydatigena and T. solium in areas where pigs share the same environmental conditions:

Braae, U.C., Kabululu, M., Nørmark, M.E., Nejsum, P., Ngowi, H.A. & Johansen, M.V. 2015. Taenia hydatigena cysticercosis in slaughtered pigs, goats, and sheep in Tanzania. Tropical Animal Health and Production. 47(8):1523–1530.

Chembensofu, M., Mwape, K.E., Van Damme, I., Hobbs, E., Phiri, I.K., Masuku, M., Zulu, G., Colston, A., et al. 2017. Re-visiting the detection of porcine cysticercosis based on full carcass dissections of naturally Taenia solium infected pigs. Parasites and Vectors. 10(1):1–9.

Delano, M.L., Mischler, S.A. & Underwood, W.J. 2002. Biology and Diseases of Ruminants : Second Edition ed. Elsevier Inc.

Dermauw, V., Ganaba, R., Cissé, A., Ouedraogo, B., Millogo, A., Tarnagda, Z., Hul, A. Van, Gabriël, S., et al. 2016. Taenia hydatigena in pigs in Burkina Faso: A cross-sectional abattoir study. Veterinary Parasitology. 230:9–13.

Gemmell, M.A. 1976. Factors regulating tapeworm populations: estimations of the build-up and dispersion patterns of eggs after the introduction of dogs infected with Taenia hydatigena. Research in Veterinary Science.

Comments: Lines 288-291. It is incorrect to say that they would need carcass dissection plus PCR to differentiate animals infected with T. solium and T. hydatigena. For the vast majority of infections with either of these two parasites the identity of the species involved is clear based on the cysticercus morphology and site of encystment. It is recommended that the words ‘coupled with PCR’ be deleted.

Response: This has been noted and revised accordingly. Please note that Chembensofu et al. 2017 made the following observation: “An important finding from our study is the presence of T. solium cysticerci in the liver, spleen and lungs, locations that are not routinely included in full carcass dissections.” Hepatic T. solium cysticerci are not always easily differentiated from juvenile T. hydatigena cysticerci, hence the need for molecular confirmation.

Comments: Lines 296-298. Care needs to be exercised in commenting on the results of other publications which involved use of serology for determining the prevalence of T. solium. For many years the Ag-ELISA test was used and the fact that the test is not species specific was virtually ignored and the results assumed to relate to T. solium. Recent detailed evaluations of the test’s performance have revealed that there is a high proportion of animals that are positive in the test that have neither T. solium nor T. hydatigena infected. For example, Chembensofu et al 2017 (DOI 10.1186/s13071-017-2520-y) found that from 30 pig carcasses that were fully dissected and found negative for T. solium, the Ag-ELISA returned ten positives, of which two were infected with T. hydatigena. That is 8/30 (26.6%) animals that had neither T. solium nor T. hydatigena infection tested false positive in Ag-ELISA. With a false positive rate of this magnitude, it is not inconceivable that all 9 Ag-ELISA positive animals from the 126 that were tested in the study described in this manuscript may have no Taenia infection at all…

Response: Noted. The authors agree with the reviewer that cross-reactivity between T. hydatigena and T. solium remains a challenge. The authors discussed this in more details in the introduction and discussion sections of the manuscript. In the same study cited by the reviewer (Chembensofu et al 2017), the authors indicated that the sensitivity of the Ag-ELISA to detect carcasses with viable cysticerci (1 or more) was estimated at 91% (95% CI: 71–99%), and increased further to 100% (95% CI: 75–100%) for carcasses with at least 10 viable cysticerci. Therefore, the validity of the results of this study still remains as the objective was to investigate the prevalence of T. solium but prevalence of Taenia species. We agree that the study of Chembensofu et al. 2017 would indicate -the existence of ‘transient’ antigens. It is indeed possible that the Ag-ELISA detects infections at the early stages of infection (Deckers et al., 2008), before establishment of cysticerci, and that in some cases infections are ‘aborted’, i.e. that infection do not result in establishment of cysticerci because of immune-driven interruption, of the infection.

Deckers N., Kanobana K., Silva M., Gonzalez A.E., Garcia H.H.,Gilman R.H., Dorny P. (2008). Serological responses in porcine cysticercosis:A link with the parasitological outcome of infection. International Journal for Parasitology 38, 1191–1198

Comments: Lines 352-353. The sharing of the environment of children and pigs with dogs has nothing whatsoever to do with transmission of T. solium.

Response: This has been noted and revised accordingly.

Comments: Lines 400-401. It is difficult to understand which authors did what – it appears that the letters refer to authors’ Christian names. It is suggested to use all the authors’ initials.

Response: This has been noted and revised accordingly.

Reviewer 2

Comments: Somewhere, in the introduction (lines 64 – 66) and in the discussion (lines 280-283) the authors clearly indicate that similar studies have been done in the same area. In my opinion, results of an undertaking reported here are just a duplication of something which has already been reported and doesn’t add any value.

Response: The authors note the concerns raised by the reviewer and have revised the manuscript accordingly. Please see below.

“There is evidence of Taenia spp. circulating in pigs in Gauteng, with 14% pigs reported to be infected in selected areas [15]. In 2016 reports of illegally slaughtered pigs for human consumption and high number of pigs with possible T. solium infection from the areas under study surfaced. Furthermore, pig farmers in these areas were reported to have had poor husbandry practices and the farms were in close proximity to human settlements. In view of this, this study estimated the seroprevalence of active Taenia spp. in domestic pigs in the selected areas of Gauteng and identified factors associated with infections and transmission based on knowledge and farming practices of pig owners. We hypothesize that Taenia spp. are circulating among pigs in township areas under study and that factors associated with the burden of Taenia spp. exist in these areas. “

TITLE

Comments: The authors need to recast the title, they could write it better than the way it is currently

Response: The title has been revised to read as “Knowledge, practices and seroprevalence of Taenia species in smallholder farms in Gauteng, South Africa”

ABSTRACT

Comments: Line 15-16: The sentence “Poor agricultural practices, and sanitary practices, and lack of…………” should be re-written to reduce the number of “ands”

Response: Changes made

Comments: Line 22-25: The sentence “Descriptive statistics were run, and logistic regression models were used to assess the relationship between socio demographic and pig farming conditions and knowledge of porcine cysticercosis and neurocysticercosis.” should be re-written to reduce the number of “ands”

Response: The sentence has been rephrased to read as “Logistic regression models were used to assess the relationship between predictor variables and the outcome variables knowledge of porcine cysticercosis or knowledge of neurocysticercosis.”

INTRODUCTION

Comments: Lines 64 – 66: the authors could not clearly indicate the rationale of conducting the study

Response: The rationale of the study has been revised to read as “There is evidence of Taenia spp. circulating in pigs in Gauteng, with 14% pigs reported to be infected in selected areas [15]. In 2016, reports of illegally slaughtered pigs for human consumption and high number of pigs with possible T. solium infection from areas under study surfaced. Furthermore, pig farmers in these areas were reported to have had poor husbandry practices and the farms were in close proximity to human settlements. In view of this, this study estimated the seroprevalence of active Taenia spp. in domestic pigs in the selected areas of Gauteng and identified factors associated with infections and transmission based on knowledge and farming practices of pig owners. We hypothesize that Taenia spp. are circulating among pigs in township areas under study and that factors associated with the burden of Taenia spp. exist in these areas.”

MATERIALS AND METHODS

Comments: In line 86 the authors indicate the use of mixed method which are not evident in the subsequent part of the study design.

Response: We thank the reviewer for the comments. There is evidence of both qualitative and quantitative approach in this study. The selection of the study areas is based on the reports by veterinary extension services and the challenges they had identified in the areas understudy. Therefore, the qualitative approach formed the basis of this study. Furthermore, both positivism and non-positivism approach although not explicitly outlined there are in line with the approach used by the authors. The positivism approach refers to quantifying the level of Taenia species in pigs based on the serological tests. The non-positivism is in the knowledge and practises of pig farmers in relation to cysticercosis and taeniasis.

Kaur, M., 2016. Application of mixed method approach in public health research. Indian Journal of Community Medicine: Official Publication of Indian Association of Preventive & Social Medicine, 41(2), p.93.

Bryman, A., 2006. Integrating quantitative and qualitative research: how is it done? Qualitative research, 6(1), pp.97-113.

Palinkas, L.A., Aarons, G.A., Horwitz, S., Chamberlain, P., Hurlburt, M. and Landsverk, J., 2011. Mixed method designs in implementation research. Administration and Policy in Mental Health and Mental Health Services Research, 38(1), pp.44-53.

Comments: In line 86-87 the authors state that they used purposive sampling to identify farms and farmers to participate in this study, they should clearly explain/reason justification for this.

Response: We thank the reviewer for the comments. It is possible that the reviewer might have missed this, the reasons for this approach are outlined in the study area and study populations section of the manuscript. “The selection of this type of sample is based on the elements possessing one or more attributes such as, known exposure to a risk factor or a specific disease status. This approach is often used in observational analytic studies. If a random sample is drawn from all sampling units meeting the study criteria, then it becomes a probability sample from the subset of the target population.” Dohoo, I.R., Martin, W. and Stryhn, H.E., 2003. Veterinary epidemiologic research.

Please see below

Study Area

Response: These areas were selected based on reports by veterinary extension services of poor pig farming husbandry practices and proximity between farms and human settlements.

Study population

Response: All pigs within the selected farms were tested provided that they met the following criteria i) Not pregnant, ii) older than 6 months, and iii) apparently healthy, therefore, making this step a probability sample [34]. The farmers were selected on the basis of known informal pig farming system and known risk factors for T. solium [27]. A total of 56 farmers were approached through communication with the State Veterinarians and Animal Health Technicians (AHT) from the Department of Agriculture and Rural Development in Gauteng Province. The inclusion criteria for the farmers were as follows: i) must agree to be part of the study, ii) must be 18 years or older, and iii) must be involved in pig farming as an owner or employee.

Comments: Line 87-90: the authors explain the selection procedure of pigs to be involved in the study and they eventually refer to the step as being probability, THIS IS SERIOUS.

Response: “The selection of this type of sample is based on the elements possessing one or more attributes such as known exposure to a risk factor or a specific disease status. This approach is often used in observational analytic studies. If a random sample is drawn from all sampling units meeting the study criteria, then it becomes a probability sample from the subset of the target population.” Dohoo, I.R., Martin, W. and Stryhn, H.E., 2003. Veterinary epidemiologic research.

Comments: Line 87-88: the authors indicate that all pigs that met criteria in all the selected farms were sampled, what was the essence of calculating the sample size then?

The sample size was calculated in order to have an estimate of the number of animals to be sampled provided that the population size was large enough. At the time of the design of the study, information on pig populations in township areas under study was not available.

Comments: The authors also need to state the number of farms from which pigs were sampled;

Response: The reviewer might have missed it, it is highlighted in line 88.

Comments: Line 90-95: the authors explain the selection procedure of farmers. It is not clear whether these were the owners of the farms where pigs were sampled or otherwise.

Response: The reviewer might have missed it, it is highlighted in line 88.

Comments: Line 98: the authors indicate that the calculated sample size was 185 pigs, but in Line 101: they indicate they sampled 126 pigs, this needs explanation.

Response: Although this was the estimated sample size, only 126 pigs met the criteria stated in line 90-91.

Comments: Lines 103-114: The authors do not consider “Blood collection and processing” and “Serological analysis” as part of data collection, can they give a reason to this?

Response: This has been corrected based in line with the comments of the reviewer.

Comments: Lines 128-131: The pretesting of the questionnaire doesn’t seem to make sense as the population to which it was tested is very different in characteristics from farmers. The authors need to defend their decision.

Response: As stated in line 145-149, pre-testing included all individuals that were representative of the target population with input from experts in the field.

Comments: Line 150: The sentence “The open-ended questions were analyzed using thematic analysis” should be cancelled.

Response: Change made

RESULTS

Comments: Generally, this section is poorly organized, the flow is not good

Response: We thank the reviewer for the comments. However, it would have been valuable if the reviewer had provided guidance to which part of the results the authors need to address.

In several occasions there is double presentation of results in text and in table

Response: The text provides a summary of the important findings from the results of the study. We would appreciate if the reviewer could provide guidance to which part of the results were repeated so that we are able to correct them.

DISCUSSION

Comments: Generally, the discussion is poorly organized

Response: We thank the reviewer for the comments. However, it would have been valuable if the reviewer had provided guidance to which part of the discussion the authors need to attend. We look forward to this guidance from the reviewer.

Comments: References “7” cited in line 280 and reference “36” cited in line 283 indicate that similar works were conducted in the same area. This is a weakness as resources devoted in this study could be deployed elsewhere

Response: The authors note the concerns raised by the reviewer and have revised the manuscript accordingly. Please see below. There is evidence of Taenia spp. circulating in pigs in Gauteng, with 14% pigs reported to be infected in selected areas [15]. In 2016 reports of illegally slaughtered pigs for human consumption and high number of pigs with possible T. solium infection from areas under study surfaced. Furthermore, pig farmers in these areas were reported to have had poor husbandry practices and the farms were in close proximity to human settlements. In view of this, this study estimated the seroprevalence of active Taenia spp. in domestic pigs in the selected areas of Gauteng and identified factors associated with infections and transmission based on knowledge and farming practices of pig owners. We hypothesize that Taenia spp. are circulating among pigs in township areas under study and that factors associated with the burden of Taenia spp. exist in these areas.

CONCLUSIONS

Comments: The third word in line 385 (training) does sound good to me as it is linked to imparting farmers knowledge on epidemiology of the parasite

Response: We note the comments of the reviewer.

Ethical considerations

Comments: Line 389-390: Check this “The Animal Ethics Committee Research Ethics Committee”

Response: Correction made

Comments: Line 393-394: I am doubting on the statement that; “Signed informed consent was obtained from each respondent before the questionnaires were administered”. This implies that all farmers could read and write

Response: All farmers could sign, and the study was explained in their language of comprehension before conducting the study.

Acknowledgements

Comments: Line 396: The statement “The author would like to acknowledge the Gauteng……….” Indicates that this manuscript is single authored

Response: Correction made

Reviewer 3

Comments: All the manuscript always refers to Taenia spp, but since all the study was performed in pigs and since it also states human neurocysticercosis, I recommend to change spp for solium, mainly because it is more accurate even though the species was not identified but Taenia saginata is only found in cattle and it does not cause neurocysticercosis in humans and using T. solium more precise in educational terms. An exception is lines 290-291

Response: We thank the reviewer for the comments; however, we prefer to leave the title as is due to the fact that the test used in this study does not differentiate T. hydatigena from T. solium

Comments: The authors should include the concept of KAP because they use it but it is not included as a specific issue such as in lines 117, 127, 157.

Response: Changes have been made in the introduction section of the manuscript in line with the comments of the reviewer. Please see below

“Similarly, poor sanitary and hygiene conditions and free-range systems have been associated with increased risk of Taenia spp. in pig populations [4,21-23]. In addition, the education level [24,25], knowledge on livestock management [26,27], and poor farming practices have been associated with high prevalence of Taenia spp. infection in pig populations [29,57].”

Comments: The reference to the statistic test cited on lines 182-183 should be provided.

Response: Noted. Addressed

Comments: Line 210 does not state pigs in numbers between 10 and 20. The authors should add: 11-20 pigs, 48%

Response: Noted. Addressed

Comments: Line 228, if not plain water, what other source/type of water did the farmers use?

Response: The farmers used plain water; correction has been made.

Comments: Table 3. farming purpose “all three” should be moved two lines below

Response: Noted. Addressed

Line 310 eliminate “of age”

Response: Noted. Addressed

Comments: The subject of KAP should also be included in the paragraph that starts on line 359

Response: It is not clear what the reviewer is requesting. Once we have received guide, we will make the changes as requested.

Comments: In acknowledgments I would add to the farmers that participated in the study

Response: Noted. Addressed

15 Sep 2020

PONE-D-20-14308R1

Knowledge, practices and seroprevalence of Taenia species in smallholder farms in Gauteng, South Africa

PLOS ONE

Dear Dr. Qekwana,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

Many thanks for submitting your manuscript to PLOS One

It was reviewed by the same experts in the field, and they have requested some more minor changes be made prior to acceptance.

If you could make these changes and write a response to reviewers, that will greatly expedite revision upon resubmission

I wish you the best of luck with your changes

Hope you are keeping safe and well in these difficult times

Thanks

Simon

==============================

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Reviewer #1: I am disappointed with the authors' responses to several of the comments I made on the manuscript. In many places they choose to argue, in effect, that I was wrong. This would be entirely acceptable if they were correct and I was not, but when the opposite is the case, it is galling.

For example, in response to my indicating that the value of the manuscript would have been enhanced had they purchased the serologically positive pigs and shown that they were actually infected with T. solium, the authors simply provide a list of excuses as to why they did not do it. The fact they did not do it is the only thing that is relevant, not why they could not, or choose not.

In response to my criticism about the lack of specificity of the serological test uses, the authors respond by citing a reference that specifies the test had a specificity of 67% for T. solium. That is exactly my point. That is a very poor specificity, and there is certainly a possibility that all 9 of the 126 animals that the authors found positive may well have had no T. solium infection at all. As the authors choose to cite two papers about the performance of the B158/B60 Ag-ELISA in relation to diagnosis of T. solium infection in pigs, and recognise that the test is also positive in pigs infected with T. hydatigena, for balance they should add the test’s sensitivity for diagnosis of T. hydatigena infection. This information is in the literature and I believe they will find that the test’s performance for T. hydatigena is just a good as it is for T. solium.

The authors’ response to my statement that the commercial test they used is not the same as the in-house test they cite in relation to the test’s performance, they firstly accept this is true but make excuses about what they perceive as small differences in the methodology used in the kit form of the test. They are not small differences, and they certainly have potential to affect the test’s performance.

The authors’ response to my comment that it is not necessary to invoke the use of PCR to differentiate T. solium and T. hydatigena cysts was to cite Chembensofu et al 2017 who claimed to have found numerous instances of T. solium cysts in the liver and lungs of pigs in Zambia and that Chembensofu et al validated their results by PCR. It is interesting to note that Chembensofu et al provided no actual data to support their extraordinary claims about cysts in ‘unusual’ locations, nor any information about what controls were used in their PCRs. The authors of the present manuscript seem unaware of the publication by Gauci et al 2019 https://doi.org/10.1371/journal.pntd.0007408 who, in response to the Chembensofu et al publication, investigated ‘lesions’ in the tissues of pigs from T. solium endemic areas of Nepal and Uganda, such as the liver and lung, that could possibly have been confused with being caused by T. solium. No T. solium cyst was found in any organ other than in the striated muscles and the brain. Numerous ‘lesions’ in other body organs were confirmed as being caused by a T. hydatigena or nematodes, as well as other causes. Gauci et al presented actual PCR results, and specified that tissue from the same animal and organ where a suspect lesion was found, but containing no lesion, was used as a negative control in the PCRs. I make two points here. Firstly, by statement that PCR is not required to differentiate T. solium and T. hydatigena cysts stands as correct. Secondly, it is annoying that the authors choose to argue against the point I made in my original review in relation to this matter, when they are clearly ignorant of the recent relevant literature. This is especially surprising because the two Belgian-based authors have a long history in the T. solium field.

The changes that have been made to the manuscript are adequate in relation to the (lack of) specificity of the serological studies they performed and the limitations this imposes on the interpretation of their results. I reiterate what I said in my original review, which was that the study would have been so much more meaningful if they had obtained actual evidence that any of the pigs were infected with T. solium. The authors’ reliance on poor quality serology was a major flaw in the study design and has resulted in a substantial reduction in the potential value of the study.

Reviewer #3: (No Response)

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27 Nov 2020

Comment 1: I am disappointed with the authors' responses to several of the comments I made on the manuscript. In many places they choose to argue, in effect, that I was wrong. This would be entirely acceptable if they were correct and I was not, but when the opposite is the case, it is galling.

For example, in response to my indicating that the value of the manuscript would have been enhanced had they purchased the serologically viable pigs and shown that they were actually infected with T. solium, the authors simply provide a list of excuses as to why they did not do it. The fact that they did not do it is the only thing that is relevant, not why they could not or choose not.

Response: The authors sincerely apologize for the miscommunication. It was not our intention to be argumentative, but our intentions were to highlight the challenges we phased during the implementation of the study. We concur that the quality of this study would have been enhanced if serologically viable pigs were purchased and the body parts excised to confirm the infection using PCR-RFLP. In trying to address the concern of the reviewer we have highlighted these limitations in lines 376 to 381.

Comment 2: In response to my criticism about the lack of specificity of the serological test uses, the authors respond by citing a reference that specifies the test had a specificity of 67% for T. solium. That is exactly my point. That is a very poor specificity, and there is certainly a possibility that all 9 of the 126 animals that the authors found positive may well have had no T. solium infection at all. As the authors choose to cite two papers about the performance of the B158/B60 Ag-ELISA in relation to diagnosis of T. solium infection in pigs, and recognise that the test is also positive in pigs infected with T. hydatigena, for balance they should add the test’s sensitivity for diagnosis of T. hydatigena infection. This information is in the literature and I believe they will find that the test’s performance for T. hydatigena is just a good as it is for T. solium.

Response: The serological assay used (B158/B60 Ag-ELISA) detects circulating antigens of Taenia spp. which indicates infection with either Taenia hydatigena or Taenia solium. For this reason, one of the aims of this paper was to study the seroprevalence of Taenia spp., without specifying. In the Chembensofu et al 2017 paper, the specificity (se) and sensitivity (sp) of the Ag-ELISA for the diagnosis of T. solium were estimated at 67% and 68%, respectively, while the sp and se of this test for diagnosing T. hydatigena infection were 49% and 86%, respectively when considering the results of the 68 dissected pigs. We agree with the reviewer that it cannot be excluded that all of the 9 of the 126 animals that were found positive by Ag-ELISA were infected with T. hydatigena.

Comment 3: The authors’ response to my statement that the commercial test they used is not the same as the in-house test they cite in relation to the test’s performance, they firstly accept this is true but make excuses about what they perceive as small differences in the methodology used in the kit form of the test. They are not small differences, and they certainly have potential to affect the test’s performance.

Response: Thank you for this remark. There are indeed differences in test conditions between the in house and the commercial Ag-ELISA. The commercial test has reduced the number of steps in the ELISA, modified the substrate and uses a different method for cut off calculation, in order to make the test more user friendly. Nevertheless, one of the authors of this paper was involved in the validation of the commercial test, during which the results of a substantial number of well-documented serum samples were compared with those of the in house test and test performance was not significantly different between the two test formats.

Comment 4: The authors’ response to my comment that it is not necessary to invoke the use of PCR to differentiate T. solium and T. hydatigena cysts was to cite Chembensofu et al 2017 who claimed to have found numerous instances of T. solium cysts in the liver and lungs of pigs in Zambia and that Chembensofu et al validated their results by PCR. It is interesting to note that Chembensofu et al provided no actual data to support their extraordinary claims about cysts in ‘unusual’ locations, nor any information about what controls were used in their PCRs. The authors of the present manuscript seem unaware of the publication by Gauci et al 2019 https://doi.org/10.1371/journal.pntd.0007408 who, in response to the Chembensofu et al publication, investigated ‘lesions’ in the tissues of pigs from T. solium endemic areas of Nepal and Uganda, such as the liver and lung, that could possibly have been confused with being caused by T. solium. No T. solium cyst was found in any organ other than in the striated muscles and the brain. Numerous ‘lesions’ in other body organs were confirmed as being caused by a T. hydatigena or nematodes, as well as other causes. Gauci et al presented actual PCR results and specified that tissue from the same animal and organ where a suspect lesion was found, but containing no lesion, was used as a negative control in the PCRs. I make two points here. Firstly, by statement that PCR is not required to differentiate T. solium and T. hydatigena cysts stands as correct. Secondly, it is annoying that the authors choose to argue against the point I made in my original review in relation to this matter, when they are clearly ignorant of the recent relevant literature. This is especially surprising because the two Belgian-based authors have a long history in the T. solium field.

Response: The authors are aware of the paper of Gauci et al. 2019, and it is indeed interesting to observe these contrasting results. We agree that some studies, including the Gauci et al 2019 study have performed dissection and have not found cysts in other organs except for those routinely examined for T. solium infections such as striated muscles and brain. However, the presence of T. solium cysticerci in the liver has also been found in pigs in Burkina Faso (Dermauw et al 2016 doi: 10.1016/j.vetpar.2016.10.022). Two of the authors of the present paper were involved in the pig dissections of either the Dermauw et al. or in both the Dermauw et al. and the Chembensofu et al papers.

In both the Dermauw and Chembensofu studies, suspected lesions were excised from the liver and other organs and transferred to individual tubes containing ethanol to avoid cross contamination of genetic material. DNA extractions and PCR’s were performed in the molecular lab of ITM Antwerp, according to SOP’s and a stringent laboratory quality system, including the use of negative and positive controls.

Following the findings of T. solium cysticerci in the liver and other organs, we consider the confirmation of suspected lesions by molecular methods important in the validation or identification of Taenia spp. cysts to nullify dubious cysts and confirm suspected lesions.

Comment 5: The changes that have been made to the manuscript are adequate in relation to the (lack of) specificity of the serological studies they performed and the limitations this imposes on the interpretation of their results. I reiterate what I said in my original review, which was that the study would have been so much more meaningful if they had obtained actual evidence that any of the pigs were infected with T. solium. The authors’ reliance on poor quality serology was a major flaw in the study design and has resulted in a substantial reduction in the potential value of the study.

Response: Thank you for your appreciation of the implemented changes. With regard to the comment on the use of serology, kindly refer to our response on Comment 1.

3 Dec 2020

Knowledge, practices and seroprevalence of Taenia species in smallholder farms in Gauteng, South Africa

PONE-D-20-14308R2

Dear Dr. Qekwana,

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Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Simon Clegg, PhD

Academic Editor

PLOS ONE

Additional Editor Comments:

Many thanks for resubmitting your manuscript to PLOS One

As you have addressed all the comments, and the manuscript reads well, I have recommended the manuscript for publication

You should hear from the Editorial office soon

It was a pleasure working with you, and I wish you the best of luck for your future research

Hope you are keeping safe and well in these difficult times

Thanks

Simon

9 Dec 2020

PONE-D-20-14308R2

Knowledge, practices and seroprevalence of Taeniaspecies in smallholder farms in Gauteng, South Africa

Dear Dr. Qekwana:

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