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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 4  |  Issue : 1  |  Page : 12-16

Seroprevalence of Toxocara canis and the parasitic effect on plasma cytokines in children aged 6 to 11 years in Saki-East local government area in Nigeria


1 Department of Medical Laboratory Science, Edo University, Iyamho, Nigeria
2 Department of Education, Medical Laboratory Science Council of Nigeria, Abuja, Nigeria

Date of Submission14-Dec-2018
Date of Acceptance26-Feb-2019
Date of Web Publication9-Apr-2019

Correspondence Address:
Prof. Mathew Folaranmi Olaniyan
Department of Medical Laboratory Science, Edo University, Iyamho
Nigeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ed.ed_23_18

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  Abstract 


Study Background: Toxocara canis is a helminths parasite known as dog worm but infects human through infected dog feces or contaminated dirt. It is a neglected disease in economically less privileged areas. The parasitic infection can generate inflammatory process.
Aim and Objective: This work was designed to determine the seroprevalence of T. canis and the parasitic effect on plasma cytokines in children aged 6–11 years in Saki-East local government area in Nigeria.
Materials and Methods: Two hundred children aged 6–11 years (female – 100 and male – 100) were recruited across Saki-East local government area. One hundred and sixty-one of them not infected with any of the infectious agents were studied as control. Sputum, blood, stool, skin snip, and urine samples were obtained for microscopic identification of parasites. Sputum sample was also used for Ziehl–Neelsen staining to demonstrate acid-fast bacilli, while ELISA technique was used for determination of T. canis, anti-hepatitis C virus (HCV), HBsAg, HIV1-p24 antigen, tumor necrosis factor-alpha (TNFα), and interleukin (IL)-10.
Results: The results obtained showed overall seroprevalence of T. canis among the children as 9.5% (19) including 6% (12) male and 3.5% (7) female children. This included 5.5% (11) monoinfected with T. canis; 1% (2) were T. canis coinfected with Ascaris lumbricoides; 1% (2) were T. canis coinfected with hepatitis B virus; 1% (2) were T. canis coinfected with Plasmodium spp.; 0.5% (1) were T. canis coinfected with hookworm; and 0.5% (1) were T. canis coinfected with Schistosoma haematobium. There is no coinfection with either HIV or HCV among the children. 10% (20) were infected with infectious agents but seronegative to T. canis. There was a significant increase in the plasma values of cytokines TNFα and IL-10 in T. canis monoinfected children compared with the control (P < 0.05).
Conclusion: The work revealed an overall seroprevalence of T. canis as 9.5% including 5.5% monoinfection and a significant increase in plasma TNFα and IL-10 in T. canis monoinfection.

Keywords: Interleukin-10, rural Nigeria, school-age children, seroprevalence, Toxocara canis tumor necrosis factor-alpha


How to cite this article:
Olaniyan MF, Azeez MM. Seroprevalence of Toxocara canis and the parasitic effect on plasma cytokines in children aged 6 to 11 years in Saki-East local government area in Nigeria. Environ Dis 2019;4:12-6

How to cite this URL:
Olaniyan MF, Azeez MM. Seroprevalence of Toxocara canis and the parasitic effect on plasma cytokines in children aged 6 to 11 years in Saki-East local government area in Nigeria. Environ Dis [serial online] 2019 [cited 2022 May 22];4:12-6. Available from: http://www.environmentmed.org/text.asp?2019/4/1/12/255737




  Introduction Top


Dog roundworms known as Toxocara canis cause toxocariasis. Toxocara eggs are found in dog feces. If the egg is ingested. The egg will hatch into larvae and travel through the bloodstream into organs and other tissues. This development can cause fever, coughing, inflammation of the liver and blindness.[1],[2] School-age children through their physical activities most often come in contact with soil and dirt. According to CDC,[3] T. canis infection is a neglected parasitic infection that results in significant illness among those who are infected and is often poorly understood by health-care providers.

Cytokines are small proteins that modulate cellular and humoral immune responses which interleukin 10 (IL-10) is an anti-inflammatory cytokine that inhibits the synthesis of pro-inflammatory cytokines and tumor necrosis-alpha which is a pro-inflammatory cytokine.[4],[5],[6]

Tumor necrosis factor-alpha (TNFα) is a pro-inflammatory cytokine responsible for systemic inflammation and is one of the cytokines that make up the acute-phase reaction.[7] Tumor necrosis factor suppresses appetite, fever, induces insulin resistance, and stimulates phagocytosis.[8]

Cytokines can be grouped into immunological cytokines that enhance cellular immune responses (e.g., TNFα) and those that favor antibody responses (e.g., IL-10). Triggers of cytokines include infectious agents, and a cytokine can trigger the release of another cytokine.[4],[5],[6]

This work was designed to determine the seroprevalence of T. canis and the parasitic effect on plasma cytokines in school-age children of a rural community in Nigeria.


  Materials and Methods Top


Study area

Saki East is a Local Government Area in Oyo State, Nigeria, with her headquarters in Ago-Amodu. It shares borders with Saki West, Atisbo, and Oorelope local governments. It has an area of 1569 km2 and a population of 110,223 according to 2006 National population census. It has five major communities which are Ago-Amodu, Sepeteri, Ogbooro, Oje-Owode, and Agbonle. There are 156 primary schools (both public and private) and 11 (11) public secondary schools and about six private schools across the Local Government area. There are a number of health centers and maternity clinic across the nook and crannies of the Local Government. It hosts a branch of the University College Hospital, Ibadan in Sepeteri.

Study population

The study population includes 200 children (female – 100 and male – 100) randomly selected across Saki-East local government area aged 6–11 years. One hundred and sixty-one of them not infected with the infectious agents were studied as control.

Methods

Human anti-Toxocara canis IgG ELISA kit was purchased from Abcam (b108775)

Principle – a 96-well plate has been precoated with T. canis antigens to bind cognate antibodies. Controls or test samples are added to the wells and incubated. Following washing, a horseradish peroxidase (HRP) labeled protein A conjugate is added to the wells, which binds to the immobilized T. canis-specific antibodies. Tetramethylbenzidine (TMB) is then catalyzed by the HRP to produce a blue color product that changes to yellow after adding an acidic stop solution. The density of yellow coloration is directly proportional to the amount of T. canis IgG sample captured in plate.

Tumor necrosis factor-alpha ELISA

Plasma TNF-alpha was determined in the participants using Abcam's kit.

A monoclonal antibody-specific TNF-alpha has been coated onto the wells of the microtiter strips provided. Samples, including standards of known TNF-alpha concentrations, control specimens, or unknowns are pipetted into these wells. During the first incubation, the standards or samples and a biotinylated monoclonal antibody specific for TNF-alpha are simultaneously incubated. After washing, the enzyme streptavidin HRP that binds the biotinylated antibody is added, incubated, and washed. A TMB substrate solution is added which acts on the bound enzyme to induce a colored reaction product. The intensity of this colored product is directly proportional to the concentration of TNF-alpha present in the samples.

Interleukin-10 ELISA

Levels of plasma IL-10 were determined in the participants using Abcam's kit.

A monoclonal antibody specific for IL-10 has been coated onto the wells of the microtiter strips provided. Samples, including standards of known IL-10 concentrations, control specimens, or unknowns are pipetted into these wells. During the first incubation, the standards or samples and a biotinylated monoclonal antibody specific for IL-10 are simultaneously incubated. After washing, the enzyme streptavidin HRP that binds the biotinylated antibody is added, incubated, and washed. A TMB substrate solution is added which acts on the bound enzyme to induce a colored reaction product. The intensity of this colored product is directly proportional to the concentration of IL-10 present in the samples.

HIV-1 p24 antigen ELISA using Zeptometrix Retrotek kit

Microwells are coated with a monoclonal antibody specific for the p24 gag gene product of HIV-1. Viral antigen in the specimen is specifically captured onto the immobilized antibody during specimen incubation. The captured antigen is then reacted with a high tittered human anti-HIV-1 antibody conjugated with biotin. Following a subsequent incubation with Streptavidin-Peroxidase, color develops as the bound enzyme reacts with the substrate. Resultant optical density is proportional to the amount of HIV-1 p24 antigen present in the specimen.

Anti-hepatitis C virus ELISA assay

This was determined using the anti-hepatitis C virus (HCV) Core Antigen-antibody (ab50288) Abcam kit.

HBsAg ELISA test

This was assayed using Diagnostic Automation/Cortez Diagnostics, INC kit by ELISA method.

The HBsAg ELISA test kit employs an antibody sandwich ELISA technique where monoclonal antibodies unique to HBsAg are precoated on polystyrene microwell strips. The plasma sample was added together with a second. After incubation and washing, to eliminate unwanted serum proteins and unbound HRP-conjugate, chromogen solutions containing TMB and urea peroxide were added to the wells. The colorless chromogens were hydrolyzed by the bound HRP conjugate to a blue-colored product. Sulfuric acid was added to stop the reaction and the blue color then turns yellow. This color intensity is directly proportional to the amount of antigen in the samples. If the blue color remains colorless, it indicates HBsAg negative.

Demonstration of acid-fast Bacilli in sputum

Ziehl–Neelsen (Zn) staining technique was used to demonstrate acid-fast bacilli (Mycobacterium species) as described by Monica.[9]

Identification of Plasmodium species Giemsa staining-thick blood film technique

Plasmodium species were determined by Giemsa staining-thick blood film technique as described by Monica.[9]

Identification of parasites in blood, skin snip, sputum, urine, and stool

This was carried out as described by Monica.[9]

Ethical consideration

Ethical approval for this work was obtained from the ethical and research committee of Baptist Medical center Saki, Nigeria (BMCS/REC/2018//4/103; dated June 22, 2018) before the commencement of this work. Informed consent was also obtained from each of the patients.

Methods of data analysis

Data were analyzed for mean, percentage, frequency, standard deviation, t-test, and probability values at 0.05 level of significance using a Statistical Package for the Social Sciences IBM SPSS, version 18 (International Business Machines Corporation (IBM), SPSS, Armonk, New York, USA).


  Results Top


The results obtained showed that 9.5% (19) (male – 6%[12] and female – 3.5% [7]) out of the 200 children investigated were infected with T. canis. This included 5.5% (11) monoinfected with T. canis; 1% (2) were T. canis coinfected with Ascaris lumbricoides; 1% (2) were T. canis coinfected with hepatitis B virus; 1% (2) were T. canis coinfected with Plasmodium spp.; 0.5% (1) were T. canis coinfected with hookworm; and 0.5% (1) were T. canis coinfected with Schistosoma haematobium. There is no coinfection with either HIV or HCV among the children. About 80.5% (161) were not infected with microbial agents investigated and as such were used as control. Nearly 10% (20) were infected with infectious agents but seronegative to T. canis[Figure 1].
Figure 1: The seroprevalence of Toxocara canis in the children

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There was a significant increase in the plasma values of TNFα and IL-10 in T. canis monoinfected children compared with the control [P < 0.05; [Table 1], [Table 2] and [Figure 2].
Table 1: Mean and standard deviation of plasma tumor necrosis factor-alpha and interleukin-10 children monoinfected with Toxocara canis

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Table 2: Comparative analysis of plasma tumor necrosis factor-alpha and interleukin-10 in children monoinfected with Toxocara canis (n=11)

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Figure 2: Comparative description of plasma tumor necrosis factor-alpha and interleukin-10 in children monoinfected with Toxocara canis. *Significant

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  Discussion Top


The findings of this work showed that 9.5% (19) (male – 6%[12] and female – 3.5% [7]) out of the 200 children investigated were infected with T. canis. This included 5.5% (11) monoinfected with T. canis; 1% (2) were T. canis coinfected with A. lumbricoides; 1% (2) were T. canis coinfected with hepatitis B virus; 1% (2) were T. canis coinfected with Plasmodium spp.; 0.5% (1) were T. canis coinfected with hookworm; and 0.5% (1) were T. canis coinfected with S. haematobium. There is no coinfection with either HIV or HCV among the children. About 80.5% (161) were not infected with microbial agents investigated and as such were used as control. Nearly 10% (20) were infected with infectious agents but seronegative to T. canis.

Humans are infected with T. canis by ingestion of embryonated ova. Ingestion of eggs from feces-contaminated materials is the most common route of infection for humans, especially children and young adults under the age of 20 years.[3] Although rare, being in contact with soil that contains infectious eggs can also cause human infection, especially handling soil with an open wound or accidentally swallowing contaminated soil, as well as eating undercooked or raw meat of an intermediate host of T. canis.[3]

The findings could be associated with the report of Neil et al.[10] that T. canis is yet another parasite that is widely distributed in economically underprivileged tropical populations as Saki-East local government, a rural area is also an economically underprivileged tropical populations.

The overall prevalence of T. canis 9.5% (19) (male – 6% [12] and female – 3.5% [7]) and that T. canis monoinfection (5.5% [11]) obtained in this work was lower than that reported by Sowemimo et al.[9] among preschool children in Osun state, Nigeria, as 18.2% in children <1 year old to a maximum of 57.6% in children aged 3 years and above. This difference could be as a result of age class difference as preschool-age children have more contact with soil or dirt than the school-age children.[11],[12]

There was a significant increase in the plasma values of TNFα and IL-10 in T. canis monoinfected children compared with the control. This finding could be associated with the fact that infection of T. canis causes inflammation of the liver.[1],[2] Inflammatory response involves release of large amount of pro-inflammatory agents such as TNFα which can, in turn, trigger the synthesis and release of anti-inflammatory cytokines to inhibit production of pro-inflammatory cytokines such as IL-10 and hence their raised plasma levels of TNFα and IL-10.[13],[14],[15]

Furthermore, infectious agents like T. canis are triggers of cytokines to stimulate the acute-phase response (TNFα)[13],[14],[15] which may be responsible for the significant finding.


  Conclusion Top


The work revealed an overall seroprevalence of T. canis as 9.5% including 5.5% monoinfection and a significant increase in plasma TNFα and IL-10 in T. canis monoinfection. Consequently, elevated plasma TNFα and IL-10 can possibly be used to suspect T. canis infection among children as a form of health-care preventive measure against mortality and morbidity rates.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Gillespie SH. The epidemiology of Toxocara canis. Parasitol Today 1988;4:180-2.  Back to cited text no. 1
    
2.
Despommier D. Toxocariasis: Clinical aspects, epidemiology, medical ecology, and molecular aspects. Clin Microbiol Rev 2003;16:265-72.  Back to cited text no. 2
    
3.
Centers for Disease Control and Prevention. Parasites – Neglected Parasitic Infections (NPIs). Centers for Disease Control and Prevention; 2017. Available from: https://www.cdc.gov/parasites/npi/index.html. [Last reviewed on 2017 Apr 13 and Last accessed on 2018 Nov 11].  Back to cited text no. 3
    
4.
Sikka G, Miller KL, Steppan J, Pandey D, Jung SM, Fraser CD 3rd, et al. Interleukin 10 knockout frail mice develop cardiac and vascular dysfunction with increased age. Exp Gerontol 2013;48:128-35.  Back to cited text no. 4
    
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Braat H, Rottiers P, Hommes DW, Huyghebaert N, Remaut E, Remon JP, et al. A phase I trial with transgenic bacteria expressing interleukin-10 in Crohn's disease. Clin Gastroenterol Hepatol 2006;4:754-9.  Back to cited text no. 5
    
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Swardfager W, Lanctôt K, Rothenburg L, Wong A, Cappell J, Herrmann N, et al. A meta-analysis of cytokines in Alzheimer's disease. Biol Psychiatry 2010;68:930-41.  Back to cited text no. 6
    
7.
Dowlati Y, Herrmann N, Swardfager W, Liu H, Sham L, Reim EK, et al. A meta-analysis of cytokines in major depression. Biol Psychiatry 2010;67:446-57.  Back to cited text no. 7
    
8.
Walsh LJ, Trinchieri G, Waldorf HA, Whitaker D, Murphy GF. Human dermal mast cells contain and release tumor necrosis factor alpha, which induces endothelial leukocyte adhesion molecule 1. Proc Natl Acad Sci U S A 1991;88:4220-4.  Back to cited text no. 8
    
9.
Monica C. District Laboratory Practice in Tropical Countries. Part 2, 2nd ed. New York: Published in the United States of America by Cambridge University Press; 2006. Available from: http://www.cambridge.org/9780521676311. [Last accessed on 2018 Nov 11].  Back to cited text no. 9
    
10.
Neil R. Lynch Kim Eddy A, Neill Hodgen Reina I, Lopez Keven J. Turner. seroprevalence of Toxocara canis infection in tropical Venezuela. In: Transactions of the Royal Society of Tropical Medicine and Hygiene. London: 1988;82; p. 275-81.  Back to cited text no. 10
    
11.
Sowemimo OA, Lee YL, Asaolu SO, Chuang TW, Akinwale OP, Badejoko BO, et al. Seroepidemiological study and associated risk factors of Toxocara canis infection among preschool children in Osun state, Nigeria. Acta Trop 2017;173:85-9.  Back to cited text no. 11
    
12.
Parise ME, Hotez PJ, Slutsker L. Neglected parasitic infections in the United States: Needs and opportunities. Am J Trop Med Hyg 2014;90:783-5.  Back to cited text no. 12
    
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Mosser DM, Zhang X. Interleukin-10: New perspectives on an old cytokine. Immunol Rev 2008;226:205-18.  Back to cited text no. 13
    
14.
Li X, Mai J, Virtue A, Yin Y, Gong R, Sha X, et al. IL-35 is a novel responsive anti-inflammatory cytokine – A new system of categorizing anti-inflammatory cytokines. PLoS One 2012;7:e33628.  Back to cited text no. 14
    
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Locksley RM, Killeen N, Lenardo MJ. The TNF and TNF receptor superfamilies: Integrating mammalian biology. Cell 2001;104:487-501.  Back to cited text no. 15
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2]



 

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