Evaluation of ELISA for detection of Trichinella antibodies in muscle juice samples of naturally infected pigs

doi:10.1016/j.vetpar.2005.05.034

Veterinary Parasitology

Volume 132, Issues 1–2, 5 September 2005, Pages 91-95

https://doi.org/10.1016/j.vetpar.2005.05.034 Get rights and content

Abstract

The performance characteristics of an ELISA test for trichinellosis in pigs applied to muscle juice was assessed using 314 samples collected from pigs located in endemic areas of Croatia. Peptic digestion was used as the reference method. The diagnostic accuracy of the two compared dilutions (1:10 and 1:100) was considered to be high because the area under the curve (AUC) index was 0.922 and 0.920 for each dilution, respectively. In this study the two graph-receiver operating characteristic (TG-ROC) analysis was used as a tool for selecting cut-off points. Sensitivity, specificity, likelihood ratios, efficiency and Youden's index were used as indices of test accuracy. The cut-off values that minimize overall misclassification cost under an assumption of 3% prevalence were calculated. Our results indicate that the ELISA applied to muscle juice is a highly accurate test and can be adapted to process a large number of samples.

Introduction

Besides methods of direct detection such as trichinelloscopy and pooled sample digestion, serodiagnostic methods, particularly ELISA, are in use for the detection of swine trichinellosis. The use of muscle juice samples and tissue fluids for the detection of specific antibodies against Trichinella have been described using experimentally infected pigs (Haralabidis et al., 1989, Kapel et al., 1998, Gamble and Patrascu, 1996). Others (Vercammen et al., 2002) have described similar tests using muscle juice from red foxes to screen for Trichinella.

The scope of our study was to define the accuracy of ELISA using a multitude of different statistical methods. These include sensitivity and specificity, positive and negative predictive value, Youden's index, and efficiency. Our goal was to determine whether ELISA performed on muscle juice could be used with confidence in post-slaughter monitoring and surveillance as a means to control the disease.

Section snippets

Samples

The muscle samples used in this study were collected from the field during the autumn and winter of 2003. A total of 141 Crura diaphragmatis muscle samples containing Trichinella muscle larvae were collected from pigs reared on private farms in endemic regions of Croatia. An additional 173 negative samples were collected at the Municipal abattoir in a non-endemic region.

Artificial digestion assay

Previously frozen muscle samples were analyzed by artificial digestion for the presence of Trichinella larvae. The individual

Results

The performance of the ELISA as measured by the AUC was very close to 1:0.922 (0.884–0.959) for dilution 1:10, and 0.921 (0.885–0.956) for dilution 1:100 (Fig. 1).

The calculated AUCs for different dilutions of muscle juice were not significantly different (p > 0.906). The upper and lower limits of the cut-off value (d₀) were read directly from the TG-ROC analysis in Fig. 2 and are shown in Table 1. Evaluating the data using the efficiency and Younden's index graph, we found that cut-off values

Discussion

Muscle juice samples can be used in the diagnosis of T. spiralis in experimentally infected pigs by latex agglutination test, ELISA and complement ELISA (Haralabidis et al., 1989). The use of muscle juice samples gave ELISA results equivalent to those from blood or serum of experimentally infected pigs (Gamble and Patrascu, 1996, Kapel et al., 1998). An advantage of using muscle juice is long term storage of samples collected for epidemiological studies and population screening tests. The aim

References (8)

H.R. Gamble et al.
Whole blood, serum and tissue fluids in an EIA for swine trichinellosis
J. Food Prot.
(1996)
H.R. Gamble et al.
International Commission on Trichinellosis: recommendations on methods for thecontrol of Trichinella in domestic and wild animals intended for human consumption
Vet. Parasitol.
(2000)
M. Greiner et al.
A modified ROC analysis for the selection of cut-off values and the definition of intermediate results of serodiagnostic tests
J. Immunol. Meth.
(1995)
M. Greiner et al.
Principles and practical application of the receiver-operating characteristic analysis for diagnostic tests
Prev. Vet. Med.
(2000)

There are more references available in the full text version of this article.

Cited by (18)

First use of tissue exudate serology to identify Toxocara spp. infection in food animals
2024, International Journal for Parasitology
Toxocara canis and Toxocara cati are globally distributed, zoonotic roundworm parasites. Human infection can have serious clinical consequences including blindness and brain disorders. In addition to ingesting environmental eggs, humans can become infected by eating infective larvae in raw or undercooked meat products. To date, no studies have assessed the prevalence of Toxocara spp. larvae in meat from animals consumed as food in the UK or assessed tissue exudates for the presence of anti-Toxocara antibodies. This study aimed to assess the potential risk to consumers eating meat products from animals infected with Toxocara spp. Tissue samples were obtained from 155 different food producing animals in the south, southwest and east of England, UK. Tissue samples (n = 226), either muscle or liver, were processed by artificial digestion followed by microscopic sediment evaluation for Toxocara spp. larvae, and tissue exudate samples (n = 141) were tested for the presence of anti-Toxocara antibodies using a commercial ELISA kit. A logistic regression model was used to compare anti-Toxocara antibody prevalence by host species, tissue type and source. While no larvae were found by microscopic examination after tissue digestion, the overall prevalence of anti-Toxocara antibodies in tissue exudates was 27.7%. By species, 35.3% of cattle (n = 34), 15.0% of sheep (n = 60), 54.6% of goats (n = 11) and 61.1% of pigs (n = 18) had anti-Toxocara antibodies. Logistic regression analysis found pigs were more likely to be positive for anti-Toxocara antibodies (odds ration (OR) = 2.89, P = 0.0786) compared with the other species sampled but only at a 10% significance level. The high prevalence of anti-Toxocara antibodies in tissue exudates suggests that exposure of food animals to this parasite is common in England. Tissue exudate serology on meat products within the human food chain could be applied in support of food safety and to identify practices that increase risks of foodborne transmission of zoonotic toxocariasis.
First detection of Trichinella spiralis in raccoon (Procyon lotor) in Germany
2022, Veterinary Parasitology: Regional Studies and Reports
Citation Excerpt :
This thesis is supported by the fact that antibodies against Trichinella spp. were detected in raccoons found to be negative for Trichinella by the artificial digestion method (Hill et al., 2008; Cybulska et al., 2020). The antigen that was used in this ELISA had been proven to be specific for Trichinella spp. (Beck et al., 2005). This suggests the need for higher sample amounts when examining raccoons for Trichinella spp. by the digestion method.
Trichinella spp. are foodborne parasites that can cause severe and potentially fatal disease in humans. Infections occur through consumption of meat containing the infectious stage (L1). In Germany the domestic cycle has been eradicated. In wild animals sporadic occurrence is observed in species such as wild boar, red foxes and raccoon dogs. The omnivore raccoon which is an invasive species in Europe is known as a potential host but has not been studied intensely regarding this parasite in Germany until now, thus resulting in a lack of knowledge about its role in the sylvatic cycle. Raccoons from the urban area of Leipzig were investigated for several pathogens including Trichinella spp. in a cooperative project. Muscle samples of 88 individuals were examined using the artificial digestion method (ADM). One animal was found positive, which is the first detection of this parasite in a raccoon in Germany.
Low levels of Trichinella spp. antibodies detected in domestic pigs at selected slaughterhouses with farm-based exposure assessment in Bulacan, Philippines
2021, Veterinary Parasitology
Citation Excerpt :
Ultimately, experimental studies show that high infectivity can be observed from pigs and rats (Pozio and Zarlenga, 2005). At present, Trichinella is common in swine livestock especially those raised in small private farms with poor sanitation and hygiene (Beck et al., 2005). On the worldwide distribution of Trichinella, Europe has the highest reported infection rates among all continents followed only by Asia (Gottstein et al., 2009; Pozio et al., 2009).
Trichinella spp. is considered as one of the most widespread food-borne zoonotic parasites globally. The disease it causes impacts human public health, pig production, and food safety. Unfortunately in the Philippines, there is still insufficient research on the presence of Trichinella among livestock. This study aims to update its status and records in the country, by verifying the presence of Trichinella spp. IgG antibodies from the selected province, Bulacan, and link its potential presence to known animal husbandry and farm practices. This study was conducted in purposively selected slaughterhouses. Pigs were randomly selected for each slaughterhouse. Blood samples were collected and serum samples were harvested from each pig samples (n = 555). Sera were tested using ELISA for the detection of Trichinella spp. IgG antibodies. For serologically positive pigs, farm-based exposure assessment was conducted to evaluate potential routes of infection. For this study, a total of 555 blood sera, wherein three blood sera were detected to be serologically positive (low prevalence of 0.54 %, 95 % CI = 0.11–1.57). Potential infection routes point towards outdoor housing management, pigs with unknown origin, pig farms presence with rodents, and pigs fed with waste as important risks. In summary, the present paper confirms that Trichinella spp. antibodies were detected in very low prevalence in Bulacan, Philippines and demonstrated the potential utilization of antibody detection as an efficient and complementary early screening tool in Trichinella detection among pigs without immediately sacrificing livestock for the sake of testing. These results merit calls for a wider screening, testing, and isolation of Trichinella spp. in pigs from other Philippine provinces.
Development of a single-plate combined indirect ELISA (CI-ELISA) for the detection of antibodies against peste-des-petits-ruminants and bluetongue viruses in goats
2015, Small Ruminant Research
Citation Excerpt :
A total of 503 goat serum samples were used for the optimization of CI-ELISA and the result was compared with commercial c-ELISA kits. The cut-off values for the CI-ELISA were determined using TG-ROC analysis which is one of the most convenient methods to quantify the diagnostic accuracy (Beck et al., 2005). The AUC indices were 0.967 and 0.995 for PPR and BT, respectively with a high statistical significance (P < 0.0001).
Peste-des-petits-ruminants and bluetongue are two of the most important viral diseases of small ruminants. In the present study, a combined indirect enzyme-linked immunosorbent assay (CI-ELISA) was developed, which can be run parallelly on the same plate for simultaneous detection of antibodies against peste-des-petits-ruminants virus and bluetongue virus. A total of 503 goat sera samples were used for developing the CI-ELISA. The relative sensitivity and specificity of CI-ELISA for detection of antibodies against PPRV were 90% and 88.8%, respectively at cut off level of 34.52% positivity. In case of BTV, 95.9% relative sensitivity and 98.6% specificity values were obtained at cut-off level of 40.42% positivity. These results indicate that the single plate CI-ELISA reported here could be used as an alternate method to existing dual plate competitive ELISAs for diagnosis/sero-surveillance of both the diseases.
Occurrence and genotypic analysis of Trichinella species in Alaska marine-associated mammals of the Bering and Chukchi seas
2014, Veterinary Parasitology
Citation Excerpt :
Forbes et al. (2003) found digestion assays three times more likely than trichinoscopy to identify positive samples, which suggests a potential for trichinoscopy-based monitoring to underestimate low-level infections (Forbes and Gajadhar, 1999; Forbes et al., 2003; Leclair et al., 2003). While enzyme-linked immunosorbent assay (ELISA) testing of serum or muscle fluid is another possible detection method (Beck et al., 2005; Gajadhar et al., 2009), the ELISA method can produce false-negatives if the animals tested were in early stages of infection (Gajadhar et al., 2009). Recently, real-time PCR was shown to be a promising option for in situ detection (Cuttell et al., 2012); however this method was not in practice at the time of our study.
The zoonotic parasite Trichinella is the causative agent of trichinellosis outbreaks in the circumpolar Arctic. Subsistence communities are particularly prone to trichinellosis due to traditional meat preparation methods and regional presence of a freeze-tolerant Trichinella species (Trichinella nativa). This study is the first application of a validated artificial digestion method in determining incidence of Trichinella sp. in Alaskan mammals. Infection incidence in pinniped species (Erignathus barbatus, Eumetopias jubatus, Odobenus rosmarus divergens, and Pusa hispida) was low, with only 1/57 ringed seals infected. Polymerase Chain Reaction assays indicate T. nativa as the only species present in northern Alaska. Analysis of an archived polar bear (Ursus maritimus) muscle sample shows freeze-tolerance and longevity for T. nativa to −20 °C for 10 years and short-term freeze resistance to −80 °C when morphology was used to determine presence of live larvae. However, larval motility suggests 0% survival. An approach that combines artificial digestion with PCR based species identification has excellent potential for Trichinella sp. detection and identification of archived tissues. Overall, Trichinella in Alaskan mammals, particularly marine mammals of subsistence importance, appears to be a minor problem. These modern diagnostic techniques provide accurate insight into the presence of Trichinella in the Alaskan marine environment.
Determination of Trichinella spiralis in pig muscles using Mid-Fourier Transform Infrared Spectroscopy (MID-FTIR) with Attenuated Total Reflectance (ATR) and Soft Independent Modeling of Class Analogy (SIMCA)
2012, Meat Science
Citation Excerpt :
Regarding larvae concentration there was no-effect on the antibody production, which means that the degree of response from pig was independent of the larvae doses. These results are in agreement with other authors' reports, which have indicated that anti-Trichinella antibodies were detected only after 21 days post-inoculation (Beck, Gasparc, Mihaljevićb, Marinculica, & Brstiloc, 2005; Gamble, 1996; Ribicich et al., 2000; Van der Leek, Dame, Adams, Gillis, & Littell, 1992). ELISA results were confirmed by Western Blot analysis (Table 1), where three immunogenic bands (44, 49 and 55 kDa) associated with ESP antigen from T. spiralis were obtained after 20 days post-infection.
The aim of this work was to study the feasibility of detection of Trichinella spiralis in swine meat using Middle Infrared Spectroscopy Fourier Transform with Attenuated Total Reflectance (ATR) and Soft Independent Modeling of Class Analogy (MID-FTIR-ATR-SIMCA). Five male Pigs were orally infected at different larvae concentrations (13,000, 6500, 3500, 1625, 812 larvae/pig) and after 24 weeks the animals were euthanized. Five types of muscles were studied (leg, loin, rib, masseter, and diaphragm). Results showed that MID-FTIR-ATR-SIMCA was useful to determine the presence of T. spiralis in the samples, as the interclass distance between infected and non infected muscles varied from 13.5 to 36.8. This technique was also useful to discriminate among pig muscles, where masseter showed the largest interclass distance, while rib presented the smallest one. In all cases the recognition and rejection rates were 100%, which means that the methodology is capable of accurately separating T. spiralis infected from non infected swine meat.

View all citing articles on Scopus

View full text

Article preview

Veterinary Parasitology

Abstract

Introduction

Section snippets

Samples

Artificial digestion assay

Results

Discussion

References (8)

Whole blood, serum and tissue fluids in an EIA for swine trichinellosis

J. Food Prot.

International Commission on Trichinellosis: recommendations on methods for thecontrol of Trichinella in domestic and wild animals intended for human consumption

Vet. Parasitol.

A modified ROC analysis for the selection of cut-off values and the definition of intermediate results of serodiagnostic tests

J. Immunol. Meth.

Principles and practical application of the receiver-operating characteristic analysis for diagnostic tests

Prev. Vet. Med.

Cited by (18)

First use of tissue exudate serology to identify Toxocara spp. infection in food animals

First detection of Trichinella spiralis in raccoon (Procyon lotor) in Germany

Low levels of Trichinella spp. antibodies detected in domestic pigs at selected slaughterhouses with farm-based exposure assessment in Bulacan, Philippines

Development of a single-plate combined indirect ELISA (CI-ELISA) for the detection of antibodies against peste-des-petits-ruminants and bluetongue viruses in goats

Occurrence and genotypic analysis of Trichinella species in Alaska marine-associated mammals of the Bering and Chukchi seas

Determination of Trichinella spiralis in pig muscles using Mid-Fourier Transform Infrared Spectroscopy (MID-FTIR) with Attenuated Total Reflectance (ATR) and Soft Independent Modeling of Class Analogy (SIMCA)