Accurate and rapid detection of Fasciola hepatica copro‐DNA in sheep using loop‐mediated isothermal amplification (LAMP) technique

Abstract Fascioliasis is a parasitic infection caused by Fasciola spp. in humans and animals. Despite significant advances in vaccination and new therapeutic agents, little attention has been paid to validate methods for the diagnosis of fascioliasis in animals. This study aimed to compare the loop‐mediated isothermal amplification (LAMP) technique with PCR assay for the diagnosis of F. hepatica in sheep. In this cross‐sectional study, 195 stool samples were collected from sheep for 3 months in Lorestan province, West of Iran. Specimens’ parasitological examination was performed by using the direct wet mount and formalin‐ether concentration method. After DNA extraction from the samples, molecular analysis was done using PCR and LAMP techniques based on the Fasciola ribosomal intergenic spacer (IGS) sequence. Of 195 specimens of sheep, 11 specimens were identified as F. hepatica‐positive infection by using microscopic, PCR and LAMP assays. Kappa agreement test results showed that there was a significant agreement between the results of microscopic examination diagnostic tests, PCR and LAMP (Kappa = 0.51–0.72 and p < .001). According to the results of chi‐square comparisons between parasite prevalence applying different techniques and variables of age, sex breed, and type of drinking water, there was no significant relationship (p ≥ .05). However, most of the infected sheep with Fasciola were 3‐ to 4‐year‐old females, of the Lori breed and consumed tap water. In many endemic areas, successful prevention and treatment of fascioliasis in animals depend on rapid and accurate diagnosis. Based on the results of the Kappa agreement, the significant agreement among the results of the microscopic examination, PCR and LAMP indicates the accuracy and reliability of these tests in the diagnosis of F. hepatica in sheep. However, molecular methods, especially the LAMP technique, are suggested because of their higher sensitivity and reliability for the diagnosis of F. hepatica even under field conditions.


| INTRODUC TI ON
Fasciola hepatica (F. hepatica), known as liver fluke, is a parasitic trematode which is commonly observed in temperate climates. F. hepatica not only infects sheep and cattle but also infects horses, deer, buffalo and camelids (John et al., 2019). Flukes are commonly found in the liver and bile ducts of their definitive host, and causes acute and chronic diseases with clinical signs such as anaemia, liver dysfunction and weight loss. The liver flukes F. hepatica and Fasciola gigantica (F. gigantica) are the agents causing fasciolosis, a zoonotic parasitic disease with typical clinical signs of fever, nausea, a swollen liver, skin rashes and extreme abdominal pain (John et al., 2019). Fasciola not only infects the liver and biliary ducts, but it may also infect the peritoneal cavity, lungs, subcutaneous tissue, lymph nodes, eye and other locations. Fasciolosis can induce considerable mortality and morbidity in livestock (Hosseini-Safa et al., 2019).
According to the Centers for Disease Control and Prevention (CDC) reports F. hepatica is found in focal areas of more than 70 countries, in all continents except Antarctica. It is found in parts of Latin America, Europe, the Caribbean, Africa, the Middle East, Oceania and Asia. F. gigantica is found in fewer geographic regions (CDC, 2018). Bennett and Ijpelaar (2005) estimated that F. hepatica causes an annual economic loss of £40.4 million in the UK cattle industry. The prevalence of 0.1% to 91.4% was reported in various livestock in Iran. In past decades, the infection rates of livestock were higher in southern Iran, while the human disease has been mostly reported in Northern provinces especially in Rasht (Ashrafi, 2015;Badparva et al., 2009;Fallahi et al. 2016).
Many advances are observed in vaccination against F. hepatica, but validated methods for the diagnosis of fascioliasis are still unknown.
Fascioliasis is commonly diagnosed with fecal testing and finding parasitic eggs in stool, bile or fluid in the duodenum through wet mount and/or condensation techniques such as formalin-ether and Telman or Kato-Kats assays. Some serologic techniques such as Fas2-enzymelinked immunosorbent assay (Fas2-ELISA), immunofluorescence assay (IFA) and Indirect Hemagglutination Assay (IHA) are applicable at all stages of the disease. These techniques have disadvantages such as the examiner's expertise and the number of parasite eggs in the stool sample. The different molecular techniques are used for diagnosis and study of Fasciola including whole-genome sequencing (WGS) and single nucleotide polymorphism (SNP), Random amplified polymorphic DNA (RAPD) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP; Hamoo et al., 2019). Loop-mediated isothermal amplification (LAMP) is a simple, sensitive and inexpensive assay which allows rapid and high-sensitive amplification of a small amount of DNA under isothermal conditions (typically at 63°C; Fallahi et al., 2014Fallahi et al., , 2015Kheirandish et al., 2020;Mirahmadi et al., 2020;Mori et al., 2001;Soltani Tehrani et al., 2020;Valian et al., 2020). The LAMP has a specific reaction because it uses four specific primers [viz. F3, FIP (F1c + F2), BIP (B1c + B2) and B3] binding to six independent sites in the target sequence Ghodsian et al., 2019;Hanifehpour et al., 2019;Parida et al., 2008). An appropriate selection of the target gene and the optimal design of turn-back primers have successful LAMP result (Fallahi et al., , 2018Ghodrati et al., 2017;Hoa Le et al., 2012) and cause visible turbidity (Mori & Notomi, 2009). This technique is carried out using inexpensive equipment such as a regular water bath or a heating block, and its products are easily seen with the eye or visualized by the addition of fluorescent dyes such as SYBR green I, with or without using a UV lamp (Mori et al., 2001).
LAMP is a technique for the rapid diagnosis of a range of zoonotic Platyhelminthes, such as F. hepatica and F. gigantica (Ai et al., 2010), Clonorchis sinensis , Schistosoma japonicum , Taenia spp. (Nkouawa et al., 2009), Paragonimus westermani (Chen et al., 2011) and, recently, Opistorchis viverrini (Arimatsu et al., 2012). In many endemic areas, successful prevention and treatment of fascioliasis in animals depend on rapid and accurate diagnosis. The present study aimed to apply and compare the two molecular methods, LAMP and PCR, in the diagnosis of F. hepatica in sheep stool samples.

| Direct stool examination
For parasitological examination, about 30 ml of normal saline (0.9% sodium chloride isotonic solution) was poured into the sampling container and placed at room temperature (20-22°C) for 30 min. The sample mixed with normal saline passed through the sieve to remove large stool particles from the sample. After completion of this step, the passed mixture was poured into an Eppendorf tube and centrifuged at 2,000 g for 4 min. A drop of sediment was deposited on the slide and examined directly at microscopic magnifications of 4, 10 and 40×.

| Formalin ethyl acetate concentration
A few grams of fecal sample precipitate passed through the sieve in a test tube was mixed with approximately 9 ml of 10% formalin and incubated at room temperature for 30 min, and then 2-3 ml of ethyl acetate was added to each tube. The lids were blocked and each tube was shaken for 30 s. Then, in order to remove the gas produced in the tube, the tube cap was gently removed and the samples were centrifuged at 2,500 g for 4 min. Each tube consists of four layers (bottom to top: fecal sample sediment, formalin, debris and ethyl acetate respectively). The debris layer was removed by an applicator and then all the contents of the pipe except the sediment were discarded. From the resulting precipitate, a drop was placed on the slide and examined at microscopic magnifications of 4, 10 and 40 ×.

| DNA extraction
A specific DNA extraction kit (MBST Co.) was used for extracting the DNA from stool samples according to the kit manufacturer's instructions. To break the Fasciola eggshell and facilitate the DNA extraction before extraction, the deposition of each stool sample that was passed through the sieve in the first step was sonicated at 90 volts and 5 s for five times. Then, 300 µl of the sample was poured into 1.5 µl microtubes and DNA extraction was performed. Eggs isolated from the uterus of several Fasciola worms that were isolated from the liver of infected slaughterhouse animals and identified and confirmed morphologically were used as a positive control for sonication and extraction procedures. After each step of sonication for 5 s, a drop of the sample was placed on the slide to check for egg breakage. Also, after the extraction step on the control samples, the extracted DNA concentration was measured using a Nanodrop machine (Thermo Scientific™ NanoDrop™ 2000/2000c Spectrophotometers, Thermo Fisher Scientific).

| PCR assay
The primers used in the LAMP technique targeting the ribosomal intergenic spacer (IGS) region of the F. hepatica genome were adapted from the study of Ai et al. (2010). The F3 and B3 external primers of the LAMP technique were also used in the PCR assay. PCR was conducted in a final volume of 25 μl containing 12.5 µl of master mix (Ampliqon), 2 µl of F and R primers, 2 µl of extracted DNA and 8.5 µl of distilled water. Reactions were amplified in a thermocycler (Bio-Rad) as follows: initial denaturation at 94°C for 5 min; afterward 35 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 45 s and extension at 72°C for 1 min. The final extension was performed at 72°C for 5 min. Negative-control samples (sterile water) were included in all PCRs. The PCR products were electrophoresed in a 1% TBE (Tris base-boric acid-EDTA) agarose gel and stained with DNA safe stain solution (1 μg/ml). The PCR amplification is expected to yield 220 bp products for a positive reaction.

| LAMP reaction solution and optimization
Optimization of LAMP with specific primers was conducted using three different cycling temperatures 61°C, 63°C and 65°C and two different concentrations of Mg 2+ , 8 and 10 mM. Another optimization was conducted using the same protocol but with three different amplification times, 30, 45 and 60 min, at 63°C with 8 mM Mg 2+ concentration. All LAMP products were stored at 4°C for further analyses.
The LAMP assay was carried out in a total of 25 µl reaction mix- was omitted in one reaction as the negative control. The mixture was incubated at 61-65°C for 60 min and then heated at 80°C for 10 min to terminate the reaction. The LAMP products were analysed by naked eyes using the turbidity of magnesium pyrophosphate. Also, 1 µl of 1/10 dilution of SYBR Green I (Invitrogen lot: 49743A) was added in the reaction tubes to visually inspect LAMP amplicons, and afterward fluorescent signals of the reaction mixtures were observed using UV Image system (UVItec). To avoid carryover contamination with LAMP amplicons by opening the tubes, the reaction tubes were centrifuged at 1,300 g for 3 min and then were frozen at −20°C for 10 min, before adding the SYBR Green I.

| The analytical sensitivity of the LAMP and PCR
The analytical sensitivity of LAMP and PCR assay was determined by preparing the serial numbers of F. hepatica eggs in PBS (0, 1, 5, 10, 20, 50 and 100 eggs/ml) and extracting the DNA as described above.
All experiments on the dilution series with the LAMP and PCR methods were performed in duplicate. Negative control was included in all molecular assays.

| Sequencing PCR products for final approval
The PCR products of external primers F3 and B3 were sequenced to confirm the accuracy of the PCR and the LAMP assays.

| Statistical analysis
The Kappa agreement test was used to determine the degree of agreement between the methods. Statistical analysis was done by using the Chi-square test and SPSS software version 19. A p-value less than .05 (typically ≤.05) was considered statistically significant.

| Demographic and contextual characteristics of the studied animals
In the present study, 19.5% and 13.8% of the animal samples were collected from Khorramabad and Aligodarz cities respectively. About 48.2% of sheep examined were from 50 to 99 herds. Most of the sheep studied had traditional grazing (79.5%), were female (62.6%) and in the age group of 3-4 years (39.5%). Other demographic and contextual characteristics of the studied animals are listed in Table 1.

| Direct stool examination
Of 195 stool samples collected from sheep 4 samples (2.1%) were found to be infected with Fasciola spp. eggs by parasitology assay and microscopic examination (Figure 1).

| Positive samples collection and DNA extraction
The parasites isolated from the liver were V-shaped at their ends and had a length-to-width ratio of 2.6 to 2.8, which were in the length-to-width range of F. hepatica. The DNA concentrations of egg samples harvested from the liver of the infected animals were 145-180 ng/µl and the absorbance of 260 to 280 ratios was read between 1.9 and 2.1.

| PCR assay
Electrophoresis of PCR products on 1% agarose gel stained with DNA safe stain revealed that among 195 DNA samples extracted from sheep stool samples, 7 specimens (3.6%) were diagnosed positive with F. hepatica (Figure 2).

| Sequence analysis
Final products of the seven PCR-positive samples using F3 and B3 external LAMP primers were sequenced, which showed 100% identification and 98%-99% query coverage with the IGS region of the

| LAMP reaction
After optimization, the optimum conditions for LAMP reaction with specific primers were achieved using 8 mM Mg 2+ , at 63°C for 60 min.  showed green fluorescence indicated a positive result of F. hepatica infection (Figure 3a,b).

| The analytical sensitivity of the LAMP and PCR
The analytical sensitivities of LAMP and PCR assays were evaluated against the preparation of the serial numbers of F. hepatica egg from 0 to 100 eggs/ml. The detection limits of the LAMP and PCR assays were 1 and 10 eggs of Fasciola, respectively, showing the higher sensitivity of the LAMP technique compared with PCR (Table 2).

| D ISCUSS I ON
Although the clinical and economic importance of fasciolosis has been known for centuries, existing diagnostic tests are not very suitable for diagnosing infection in animals. The choice of the type of diagnostic method is usually influenced by the purpose of the study.
The gold standard approaches and/or experimental settings have mainly been used to evaluate the diagnostic test and their limitations (Mazeri et al., 2016). Nonetheless some advances in establishing immunologic techniques, these tests lack high sensitivity and/or specificity in diagnosis. Even so, there is considerable potential for antigen detection tests, but these tests are not sufficiently evaluated in field conditions. Stool egg count and coproantigen testing have been suggested as appropriate methods for diagnosing drug efficacy/resistance. Early diagnosis of F. hepatica infection is provided by serological methods, but circulating antibodies may remain in the blood for several months after successful treatment. Therefore, serology does not always measure the current infection but only the expo- In the current study, the routine parasitology examination (direct wet mount and concentration method), PCR and LAMP techniques TA B L E 2 The analytical sensitivity of LAMP and PCR assays against the serial numbers of <em>Fasciola</em> egg from 0 to 100 eggs/ ml They also reported that in-house ES antigen ELISA showing that while being a valuable test, its sensitivity and specificity estimates are lower in the field setting than previously reported (Mazeri et al., 2016). In a study by Ai et al. (2010)

ACK N OWLED G EM ENTS
The authors would like to thank all people who helped us in this study for their kind assistance. This article is derived from the PhD of Veterinary Parasitology thesis of the First author (Thesis No. 3122),