Hostname: page-component-cd9895bd7-jkksz Total loading time: 0 Render date: 2024-12-27T11:11:20.033Z Has data issue: false hasContentIssue false
  • English
  • Français

Visceral larva migrans detection using PCR–RFLP in BALB/c mice infected with Toxocara canis

Published online by Cambridge University Press:  09 August 2019

G. Özbakış Open the ORCID record for G. Özbakış [Opens in a new window]  and
A. Doğanay
G. Özbakış*
Affiliation:
Department of Parasitology, Faculty of Veterinary Medicine, Ankara University, Ankara, Turkey
A. Doğanay
Affiliation:
Department of Parasitology, Faculty of Veterinary Medicine, Ankara University, Ankara, Turkey
*
Author for correspondence: G. Özbakış, E-mail: ozbakis@ankara.edu.tr
Get access Rights & Permissions [Opens in a new window]

Abstract

Toxocara canis is an important zoonotic roundworm distributed worldwide. The infective larvae of T. canis are one of the causes of visceral larva migrans (VLM), a clinical syndrome in humans. Diagnosing VLM is difficult, and the differential diagnosis of the larval development stage is limited. Therefore, this experimental research aimed to diagnose T. canis larvae using a molecular method, not only in liver tissue, which is the most commonly affected tissue, but also in the limb muscles, lungs and brain tissues. For this purpose, 24 BALB/c mice were infected with 1000 embryonated T. canis eggs. Necropsies were performed on the second, fourth, seventh and 14th days post-infection. While a part of the samples were digested with pepsin-HCl, the molecular method was used for the remainder of the samples to replicate the mitochondrial DNA adenosine triphosphate (ATP) synthase subunit-6 gene region of T. canis. BbsI, a restriction endonuclease, was used to determine the specificity of the amplicons obtained from Polymerase chain reaction (PCR). The detection limit for embryonated eggs was recorded. The PCR results showed that the sensitivity of the PCR analysis was 83.3% in the liver (with 88.8% accuracy), 87.5% in the lungs (with 91.6% accuracy) and 75.0% in the brain, forelimb and hindlimb muscles (with 83.3% accuracy). In all tissues, the test specificity was determined to be 100%. In this study, the molecular method was applied to only experimentally infected BALB/c mice tissues; thus, it is suggested that it can be also employed in different paratenic hosts and materials possibly infected with T. canis.

Keywords

VLMtoxocariasispepsin-HCl digestionATPase subunit-6
Type
Research Paper
Information
Journal of Helminthology , Volume 94 , 2020 , e70
Copyright
Copyright © Cambridge University Press 2019 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Abo-Shehada, MN and Herbert, IV (1984/1985) The migration of larval Toxocara canis in mice II. Post-intestinal migration in primary infections. Veterinary Parasitology 17, 75–83.Google Scholar
Akao, N (2006) Critical assessment of existing and novel model systems of toxocariasis. pp. 74–85 in Holland, CV and Smith, HV (Eds) Toxocara the enigmatic parasite. Oxfordshire, CABI Publishing.Google Scholar
Akyol, A, Bicerol, B, Ertug, S, Ertabaklar, H and Kıylıoglu, N (2007) Epilepsy and seropositivity rates of Toxocara canis and Toxoplasma gondii. Seizure 16, 233–237.Google Scholar
Archelli, S, Santillan, GI, Fonrouge, R, Céspedes, G, Burgos, L and Radman, N (2014) Toxocariasis: seroprevalence in abandoned-institutionalized children and infants. Revista Argentina de Microbiología 46(1), 3–6.Google Scholar
Berrett, AN, Erickson, LD, Gale, SD, Stone, A, Brown, BL and Hedges, DW (2017) Toxocara seroprevalence and associated risk factors in the United States. The American Journal of Tropical Medicine and Hygiene 97(6), 1846–1850.Google Scholar
Bisseru, B (1969) Studies on the liver, lung, brain and blood of experimental animals infected with Toxocara canis. Journal of Helminthology 43(3/4), 267–272.Google Scholar
Boldis, V, Ondriska, F, Spitalska, E and Reiterova, K (2015) Immunodiagnostic approaches for the detection of human toxocarosis. Experimental Parasitology 159, 252–258.Google Scholar
Borecka, A (2004) Differentiation of Toxocara spp. eggs isolated from the soil by the PCR linked RFLP method. Helminthologia 41(4), 185–187.Google Scholar
Borecka, A, Gawor, J, Niedworok, M and Sordyl, B (2008) Detection of Toxocara canis larvae by PCR in the liver of experimentally infected Mongolian gerbils (Meriones unguiculatus). Helminthologia 45(3), 147–149.Google Scholar
Burren, CH (1968) Experimental toxocariasis I. Some observations and histopathology of the migration of Toxocara canis larvae in mouse. Zeitschrift für Parasitenkunde 30, 152–161.Google Scholar
Burren, CH (1971) The distribution of Toxocara larvae in the central nervous system of the mouse. Transactions of the Royal Society of Tropical Medicine and Hygiene 65(4), 450–453.Google Scholar
Cong, W, Meng, QF, You, HL, Zhou, N, Dong, XY, Dong, W, Wang, XY, Qian, AD and Zhu, XQ (2015) Seroprevalence and risk factors of Toxocara infection among children in Shandong and Jilin provinces, China. Acta Tropica 152, 215–219.Google Scholar
de Moura, MQ, de Macedo, MRP, da Silva Terto, WD, da Costa Avila, LF, Leite, FPL, Scaini, CJ, Natália Berne Pinto, N, de Almeida, CG, Strothmann, AL, MarreiroVillela, M and Berne, MEA (2018) Detection of Toxocara canis DNA in tissues of experimentally infected mice. Acta Tropica 187, 51–56.Google Scholar
Despommier, D (2003) Toxocariasis: clinical aspects, epidemiology, medical ecology and molecular aspects. Clinical Microbiology Reviews 16(2), 265–272.Google Scholar
Ei-Shazly, AM, Abdel Baset, SM, Kamal, A, Mohammed, KA, Sakr, TI and Hammad, SM (2013) Seroprevalence of human toxocariasis (Visceral Larva Migrans). AAMJ 11(Supp 3), 16–34.Google Scholar
Fillaux, J and Magnaval, JF (2013) Laboratory diagnosis of human toxocariasis. Veterinary Parasitology 193(4), 327–336.Google Scholar
Fogt-Wyrwas, RF, Jarosz, W and Mizgajska-Wiktor, H (2007) Utilizing a polymerase chain reaction method for the detection of Toxocara canis and T. cati eggs in soil. Journal of Helminthology 81, 75–78.Google Scholar
Gasser, RB, Zhu, XQ, Hu, M, Jacobs, DE and Chilton, NB (2006) Molecular genetic characterization of members of the genus Toxocara-taxonomic, population genetic and epidemiological considerations. pp. 18–31 in Holland, CV and Smith, HV (Eds) Toxocara the enigmatic parasite. Oxfordshire, CABI Publishing.Google Scholar
Ishiwata, K, Shinohara, A, Yagi, K, Horii, Y, Tsuchiya, K and Nawa, Y (2004) Identification of tissue-embedded ascarid larvae by ribosomal DNA sequencing. Parasitology Research 92, 50–52.Google Scholar
Jacobs, DE, Zhu, X, Gasser, RB and Chilton, NB (1997) PCR-based methods for identification of potentially zoonotic ascaridoid parasites of the dog, fox and cat. Acta Tropica 68, 191–200.Google Scholar
Jarosz, W, Mizgajska-Wiktor, H, Kirwan, P, Konarski, J, Rychlicki, W and Wawrzyniak, G (2010) Developmental age, physical fitness and Toxocara seroprevalence amongst lower-secondary students living in rural areas contaminated with Toxocara eggs. Parasitology 137(1), 53–63.Google Scholar
Kaplan, M, Kalkan, A, Kuk, S, Demırdag, K, Ozden, M and Kılıc, SS (2008) Toxocara seroprevalence in schizophrenic patients in Turkey. Yonsei Medical Journal 49(2), 224–229.Google Scholar
Kassai, T (1999) Veterinary helminthology. 1st edn. Oxford, Butterworth-Heinemann.Google Scholar
Kustimur, S, Dogruman Al, F, Oguzulgen, K, Bakır, H, Maral, I, Turktas, H and Tuzun, H (2007) Toxocara seroprevalence in adults with bronchial asthma. Transactions of the Royal Society of Tropical Medicine & Hygiene 101, 270–274.Google Scholar
Lee, ACY, Schantz, PM, Kazacos, KR, Montgomery, SP and Bowman, DD (2010) Special issue: Zoonoses of people and pets in the USA. Epidemiologic and zoonotic aspects of ascarid infections in dogs and cats. Trends in Parasitology 26(4), 155–161.Google Scholar
Li, MW, Lin, RQ, Chen, HH, Sani, RA, Song, HQ and Zhu, XO (2007) PCR tools for the verification of the specific identity of ascaridoid nematodes from dogs and cats. Molecular and Cellular Probes 21, 349–354.Google Scholar
Liao, CW, Sukati, H, D'lamini, P, et al. (2010) Seroprevalence of Toxocara canis infection among children in Swaziland, southern Africa. Annals of Tropical Medicine & Parasitology 104(1), 73–80.Google Scholar
Lötsch, F, Obermüller, M, Mischlinger, J, et al. (2016) Seroprevalence of Toxocara spp. in a rural population in Central African Gabon. Parasitology International 65(6), 632–634.Google Scholar
Ollero, MD, Fenoy, S, Cuellar, C, Guillen, JL and Aguila, C (2008) Experimental toxocariosis in BALB/c mice: effect of the inoculation dose on brain and eye involvement. Acta Tropica 105, 124–130.Google Scholar
Oshima, T (1961) Standardization of techniques for infecting mice with Toxocara canis and observations on the normal migration routes of the larvae. Journal of Parasitology 47, 652–656.Google Scholar
Roberts, LS and Janovy, J (2006) Gerald D. Schmidt & Larry S. Roberts’ foundations of parasitology. International edn. New York, Mc-Graw Hill Press.Google Scholar
Sambrook, J, Fritschi, EF and Maniatis, T (1989) Molecular cloning: a laboratory manual. New York, Cold Spring Harbor Laboratory Press.Google Scholar
Schnieder, T, Laabs, EM and Welz, C (2011) Larval development of Toxocara canis in dogs. Veterinary Parasitology 175, 193–206.Google Scholar
Schoenardie, ER, Scaini, CJ, Brod, CS, Pepe, MS, Villela, MM, McBride, AJ, Borsuk, S and Berne, ME (2013) Seroprevalence of Toxocara infection in children from southern Brazil. Journal of Parasitology 99(3), 537–540.Google Scholar
Silva, MB, Amor, AL, Santos, LN, et al. (2017) Risk factors for Toxocara spp. seroprevalence and its association with atopy and asthma phenotypes in school-age children in a small town and semi-rural areas of Northeast Brazil. Acta Tropica 174, 158–164.Google Scholar
Sinha, BN (1966) The migratory behaviour of the larvae of Toxocara canis (Werner 1782) in white mice. Indian Veterinary Journal 43(12), 1101–1105.Google Scholar
Smith, H, Holland, C, Taylor, M, Magnaval, JF, Schantz, P and Maizels, R (2009) How common is human toxocariasis? Towards standardizing our knowledge. Trends in Parasitology 25(4), 182–188.Google Scholar
Sprent, JFA (1955) On the invasion of the central nervous system by nematodes. II. Invasion of the nervous system in ascariasis. Parasitology 45, 41–55.Google Scholar
Sprent, JFA (1958) Observations on the development of Toxocara canis (Werner, 1782) in the dog. Parasitology 48, 184–209.Google Scholar
Strube, C, Heuer, L and Janecek, E (2013) Toxocara spp. infections in paratenic hosts. Veterinary Parasitology 193, 375–389.Google Scholar
Wang, Z, Shibata, M, Nguyen, YTH, Hayata, Y, Nonaka, N, Maruyama, H and Yoshida, A (2018) Development of nested multiplex polymerase chain reaction (PCR) assay for the detection of Toxocara canis, Toxocara cati and Ascaris suum contamination in meat and organ meats. Parasitology International 67(5), 622–626.Google Scholar
Zibaei, M, Sadjjadi, SM and Maraghi, S (2017) The occurrence of Toxocara species in naturally infected broiler chickens revealed by molecular approaches. Journal of Helminthology 91(5), 633–636.Google Scholar