Cross-Reactions between Toxocara canis and Ascaris suum in the diagnosis of visceral larva migrans by western blotting technique

Visceral larva migrans (VLM) is a clinical syndrome caused by infection of man by Toxocara spp, the common roundworm of dogs and cats. Tissue migration of larval stages causes illness specially in children. Because larvae are difficult to detect in tissues, diagnosis is mostly based on serology. After the introduction of the enzyme-linked immunosorbent assay (ELISA) using the larval excretory-secretory antigen of T. canis (TES), the diagnosis specificity was greatly improved although cross-reactivity with other helminths are still being reported. In Brazil, diagnosis is routinely made after absorption of serum samples with Ascaris suum antigens, a nematode antigenicaly related with Ascaris lumbricoides which is a common intestinal nematode of children. In order to identify T. canis antigens that cross react to A. suum antigens we analyzed TES antigen by SDS-PAGE and Western blotting techniques. When we used serum samples from patients suspected of VLM and positive result by ELISA as well as a reference serum sample numerous bands were seen (molecular weight of 210-200 kDa, 116-97 kDa, 55-50 kDa and 35-29 kDa). Among these there is at least one band with molecular weight around 55-66 kDa that seem to be responsible for the cross-reactivity between T. canis e A. suum once it disappears when previous absorption of serum samples with A. suum antigens is performed

Download Full-text

Differentiation of Larva Migrans Caused by Baylisascaris procyonis and Toxocara Species by Western Blotting

Clinical and Vaccine Immunology ◽

10.1128/cvi.00251-09 ◽

2009 ◽

Vol 16 (11) ◽

pp. 1563-1568 ◽

Cited By ~ 19

Author(s):

Sriveny Dangoudoubiyam ◽

Kevin R. Kazacos

Keyword(s):

Western Blotting ◽

Cross Reactivity ◽

High Reactivity ◽

Larva Migrans ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Human Patient ◽

Western Blots ◽

Baylisascaris Procyonis ◽

Very High

ABSTRACT Baylisascaris procyonis and Toxocara species are two important causes of larva migrans in humans. Larva migrans caused by Toxocara spp. is well known and is diagnosed serologically by enzyme immunoassay. Over a dozen cases of larva migrans and associated eosinophilic encephalitis caused by B. procyonis have also been reported, and at least a dozen additional cases are known. An enzyme-linked immunosorbent assay (ELISA) using the excretory-secretory (ES) antigen of B. procyonis larvae is currently being used in our laboratory as an aid in the diagnosis of this infection in humans. Clinically affected individuals show very high reactivity (measured as the optical density) on this ELISA; however, a one-way cross-reactivity with Toxocara spp. has been observed. As an approach to differentiate these two infections based on serology, we performed Western blots, wherein the B. procyonis ES antigen was reacted with serum samples from individuals known to be positive for either Toxocara spp. or B. procyonis larva migrans. Western blot results showed that B. procyonis antigens of between 30 and 45 kDa were specifically identified only by the sera from individuals with Baylisascaris larva migrans, thus allowing for differentiation between the two infections. This included human patient serum samples submitted for serologic testing, as well as sera from rabbits experimentally infected with B. procyonis. When used in conjunction with the ELISA, Western blotting could be an efficient tool for diagnosis of this infection in humans.

Download Full-text

Frequency of Toxocara canis antibodies in Mexican paediatric patients with epilepsy

Journal of Helminthology ◽

10.1017/s0022149x1900083x ◽

2019 ◽

Vol 94 ◽

Author(s):

M.d.L. Caballero-García ◽

J. Simón-Salvador ◽

J.C. Hernández-Aguilar ◽

A. Reyes-Lopez ◽

B. Nogueda-Torres ◽

...

Keyword(s):

Western Blotting ◽

Toxocara Canis ◽

Cross Reactivity ◽

Larva Migrans ◽

Enzyme Linked Immunosorbent Assay ◽

Healthy Children ◽

Western Blotting Analysis ◽

Molecular Weights ◽

Blotting Analysis ◽

The Relationship

Abstract The relationship between epilepsy and the presence of visceral larva migrans caused by Toxocara canis in Mexican children remains uncertain; however, this relationship needs to be elucidated because these parasite larvae can invade the human central nervous system. Accordingly, this study aimed to determine the frequency and specificity of anti-T. canis antibodies in the sera of children with epilepsy to determine the relationship between this parasite and epilepsy. The sera samples of 214 children were examined: 111 children diagnosed with epilepsy and 103 clinically healthy children without neurological disorders. In the sera of each group, the presence and specificity of anti-T. canis and anti-Ascaris lumbricoides antibodies, as well as the cross-reactivity between them, were assessed using enzyme-linked immunosorbent assay and Western blotting analysis. Among the children with epilepsy, 25.2% exhibited seropositivity to T. canis. Cross-reactivity against the A. lumbricoides antigen was present in 46.8% of the children with epilepsy, whereas 11.7% of the children with epilepsy and anti-T. canis antibodies did not exhibit cross-reactivity against this antigen. The Western blotting analysis of the sera from the children with epilepsy demonstrated the presence of T. canis proteins, with molecular weights of 24, 35, 55, 70, 120 and 210 kDa, and A lumbricoides proteins with molecular weights of 70, 80 and 110 kDa. Our results revealed the presence of anti-T. canis antibodies in the children with epilepsy; furthermore, cross-reactivity tests with A. lumbricoides showed the importance of the presence of anti-T. canis antibodies in revealing the relationship between this parasite and epilepsy in children.

Download Full-text

Cross-reactivity between Anisakis simplex sensitization and visceral larva migrans by Toxocara canis

Acta Tropica ◽

10.1016/s0001-706x(03)00201-8 ◽

2003 ◽

Vol 89 (1) ◽

pp. 85-89 ◽

Cited By ~ 19

Author(s):

M.J. Perteguer ◽

C. Cuéllar ◽

J.L. Guillén ◽

C. Águila ◽

S. Fenoy ◽

...

Keyword(s):

Toxocara Canis ◽

Cross Reactivity ◽

Anisakis Simplex ◽

Larva Migrans ◽

Visceral Larva Migrans

Download Full-text

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

Journal of Helminthology ◽

10.1017/s0022149x19000609 ◽

2019 ◽

Vol 94 ◽

Author(s):

G. Özbakış ◽

A. Doğanay

Keyword(s):

Toxocara Canis ◽

Development Stage ◽

Clinical Syndrome ◽

Larva Migrans ◽

Pcr Analysis ◽

Visceral Larva Migrans ◽

Molecular Method ◽

Affected Tissue ◽

Test Specificity ◽

Paratenic Hosts

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.

Download Full-text

Combined Use of Indirect ELISA and Western Blotting with Recombinant Hepatocellular Carcinoma-Associated Antigen 59 Is a Potential Immunodiagnostic Tool for the Detection of Prepatent Haemonchus contortus Infection in Goat

Animals ◽

10.3390/ani9080548 ◽

2019 ◽

Vol 9 (8) ◽

pp. 548 ◽

Cited By ~ 7

Author(s):

Muhammad Ali-ul-Husnain Naqvi ◽

Sana Zahra Naqvi ◽

Muhammad Ali Memon ◽

Kalibixiati Aimulajiang ◽

Muhammad Haseeb ◽

...

Keyword(s):

Western Blot ◽

Western Blotting ◽

Haemonchus Contortus ◽

Indirect Elisa ◽

Fasciola Hepatica ◽

False Negative ◽

Cross Reactivity ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Combined Use

Haemonchus contortus is recognized as one of the important health problems in small ruminants, leading to reduced production and economic loss for farmers worldwide. Prepatent diagnosis of H. contortus infection is crucial to improve control strategies as this helminth may remove up to one-fifth of total erythrocytes and may cause anemia, edema, diarrhea, and ultimately death in young animals. In this study, one of the excretory and secretory products, rHc-HCA59, was purified and used as antigen to detect specific antibodies in H. contortus infected goats during prepatent stage of infection using indirect enzyme linked immunosorbent assay (ELISA) as screening test. All goats (n = 38) were housed indoor, experimentally infected with 8000 infective larvae (L3) of H. contortus, and serum samples were collected prior to infection and at 14th day of infection. Immunoblotting was performed to confirm the results of indirect ELISA, evaluate the cross reactivity against rHc-HCA59 in sera of most common co-infecting parasites and rectify the false negative samples. Furthermore, three different batches of rHc-HCA59 were produced to evaluate the repeatability of ELISA. No eggs were detected in feces of all goats collected at 7th and 14th day of infection but, H. contortus eggs were detected at 21 days post infection in the feces. Indirect ELISA performed in this study showed 87% sensitivity and 100% specificity. The western blot analysis confirmed immunoreactivity in serum samples which scored positive in indirect ELISA and recognized the samples as negative which had OD450 lower than negative cut-off value in indirect ELISA. Furthermore, all false negative sera (n = 5) that had OD450 value between positive and negative cut-off value in rHc-HCA59 based ELISA were clearly positive in western blot. Moreover, no cross-reactivity was detected in ELISA and western blotting against rHc-HCA59 in positive sera of Toxoplasma gondii, Fasciola hepatica, and Trichinella spiralis. The results of this study concluded that combined use of indirect ELISA and western blotting with rHc-HCA59 is a potential immunodiagnostic tool for the detection of H. contortus infection during prepatent period in goats.

Download Full-text

Recombinant Antigen-Based Enzyme-Linked Immunosorbent Assay for Diagnosis of Baylisascaris procyonis Larva Migrans

Clinical and Vaccine Immunology ◽

10.1128/cvi.00083-11 ◽

2011 ◽

Vol 18 (10) ◽

pp. 1650-1655 ◽

Cited By ~ 14

Author(s):

Sriveny Dangoudoubiyam ◽

Ramesh Vemulapalli ◽

Momar Ndao ◽

Kevin R. Kazacos

Keyword(s):

Immunosorbent Assay ◽

Cross Reactivity ◽

Larva Migrans ◽

Enzyme Linked Immunosorbent Assay ◽

Parasitic Infections ◽

Serum Samples ◽

Western Blot Assay ◽

Human Patient ◽

Content Type ◽

Baylisascaris Procyonis

ABSTRACTBaylisascarislarva migrans is an important zoonotic disease caused byBaylisascaris procyonis, the raccoon roundworm, and is being increasingly considered in the differential diagnosis of eosinophilic meningoencephalitis in children and young adults. Although aB. procyonisexcretory-secretory (BPES) antigen-based enzyme-linked immunosorbent assay (ELISA) and a Western blot assay are useful in the immunodiagnosis of this infection, cross-reactivity remains a major problem. Recently, a recombinantB. procyonisantigen, BpRAG1, was reported for use in the development of improved serological assays for the diagnosis ofBaylisascarislarva migrans. In this study, we tested a total of 384 human patient serum samples in a BpRAG1 ELISA, including samples from 20 patients with clinicalBaylisascarislarva migrans, 137 patients with other parasitic infections (8 helminth and 4 protozoan), and 227 individuals with unknown/suspected parasitic infections. A sensitivity of 85% and a specificity of 86.9% were observed with the BpRAG1 ELISA, compared to only 39.4% specificity with the BPES ELISA. In addition, the BpRAG1 ELISA had a low degree of cross-reactivity with antibodies toToxocarainfection (25%), while the BPES antigen showed 90.6% cross-reactivity. Based on these results, the BpRAG1 antigen has a high degree of sensitivity and specificity and should be very useful and reliable in the diagnosis and seroepidemiology ofBaylisascarislarva migrans by ELISA.

Download Full-text

Dogs as Sentinels for Flavivirus Exposure in Urban, Peri-Urban and Rural Hanoi, Vietnam

Viruses ◽

10.3390/v13030507 ◽

2021 ◽

Vol 13 (3) ◽

pp. 507

Author(s):

Long Pham-Thanh ◽

Thang Nguyen-Tien ◽

Ulf Magnusson ◽

Vuong Bui-Nghia ◽

Anh Bui-Ngoc ◽

...

Keyword(s):

Mosquito Control ◽

Risk Mitigation ◽

Significant Risk ◽

Cross Sectional Study ◽

Cross Reactivity ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Cross Sectional ◽

Major Health ◽

Household Level

Diseases caused by flaviviruses, including dengue fever and Japanese encephalitis, are major health problems in Vietnam. This cross-sectional study explored the feasibility of domestic dogs as sentinels to better understand risks of mosquito-borne diseases in Hanoi city. A total of 475 dogs serum samples from 221 households in six districts of Hanoi were analyzed by a competitive enzyme-linked immunosorbent assay (cELISA) for antibodies to the pr-E protein of West Nile virus and other flaviviruses due to cross-reactivity. The overall flavivirus seroprevalence in the dog population was 70.7% (95% CI = 66.4–74.8%). At the animal level, significant associations between seropositive dogs and district location, age, breed and keeping practice were determined. At the household level, the major risk factors were rural and peri-urban locations, presence of pigs, coil burning and households without mosquito-borne disease experience (p < 0.05). Mosquito control by using larvicides or electric traps could lower seropositivity, but other measures did not contribute to significant risk mitigation of flavivirus exposure in dogs. These results will support better control of mosquito-borne diseases in Hanoi, and they indicate that dogs can be used as sentinels for flavivirus exposure.

Download Full-text

Standardization of dot-ELISA for the serological diagnosis of toxocariasis and comparision of the assay with ELISA

Revista do Instituto de Medicina Tropical de São Paulo ◽

10.1590/s0036-46651992000100010 ◽

1992 ◽

Vol 34 (1) ◽

pp. 55-60 ◽

Cited By ~ 34

Author(s):

Eide Dias Camargo ◽

Paulo Mutuko Nakamura ◽

Adelaide José Vaz ◽

Marcos Vinícius da Silva ◽

Pedro Paulo Chieffi ◽

...

Keyword(s):

Low Cost ◽

Clinical Signs ◽

Toxocara Canis ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Specific Antibodies ◽

Normal Individuals ◽

Before And After ◽

Dot Elisa ◽

Stability Time

The dot-enzyme-linked immunosorbent assay (dot-ELISA) was standardized using somatic (S) and excretory-secretory (ES) antigens of Toxocara-canis for the detection of specific antibodies in 22 serum samples from children aged 1 to 15 years, with clinical signs of toxocariasis. Fourteen serum samples from apparently normal individuals and 28 sera from patients with other pathologies were used as controls. All samples were used before and after absorption with Ascaris suum extract. When the results were evaluated in comparison with ELISA, the two tests were found to have similar sensitivity, but dot-ELISA was found to be more specific in the presence of the two antigens studied. Dot-ELISA proved to be effective for the diagnosis of human toxocariasis, presenting advantages in terms of yield, stability, time and ease of execution and low cost.

Download Full-text

Diagnosis of alveolar echinococcosis using immunoblotting with plural low molecular weight antigens

Journal of Helminthology ◽

10.1017/s0022149x08116510 ◽

2009 ◽

Vol 83 (1) ◽

pp. 57-61 ◽

Cited By ~ 7

Author(s):

K. Yamano ◽

A. Goto ◽

M. Miyoshi ◽

K. Furuya ◽

Y. Sawada ◽

...

Keyword(s):

Molecular Weight ◽

Alveolar Echinococcosis ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Serological Screening ◽

Screening Process ◽

Medical Practitioners ◽

Specific Reactions ◽

Hospital Outpatients ◽

Higher Sensitivity

AbstractAlveolar echinococcosis (AE) is endemic to Hokkaido, Japan. For the past 20 years, detection of AE among inhabitants has involved serological screening using an enzyme-linked immunosorbent assay (ELISA) followed by Western blotting (WB). Between the years 1987 and 2000, antigens targeted on 66, 55 and 30–35 kDa bands were routinely used in the WB step of AE diagnosis. However, since 2001 diagnosis has been dependent on three smaller molecular weight antigens (26–28, 18 and 7–8 kDa). Due to its higher sensitivity, this improved WB approach has been used as a confirmation step in the screening process and also for the testing of suspected AE cases in hospital outpatients. Using the improved WB technique, a total of 1745 serum samples were examined in 2001–2006 with 81 patients detected and registered with AE. Interestingly, sera from 76 of the 81 diagnosed AE patients (93.8%) demonstrated reactivity with all three antigens. However, sera from the remaining five patients (6.2%) demonstrated no reactivity with the 18 kDa antigen, even though they exhibited clearly detectable levels of reactivity with the 26–28 and 7–8 kDa bands. These results suggest that medical practitioners need to pay particular attention to the specific reactions to some different diagnostic antigens to minimize the risk of misdiagnosing AE patients. In turn, these results may also provide important diagnostic information for cystic echinococcosis (CE).

Download Full-text