Veterinary Parasitology 274 (2019) 108922 Contents lists available at ScienceDirect Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar Research paper Development of Cytochrome B, a new candidate gene for a high accuracy detection of Fasciola eggs in fecal specimens T ⁎ Thapana Chontananartha,b, , Janjura Parawata a Applied Parasitology Research Laboratory, Department of Biology, Faculty of Science, Srinakharinwirot University, Bangkok, Thailand b Center of Excellence in Animal, Plant and Parasitic Biotechnology, Srinakharinwirot University, Bangkok, Thailand ARTICLE INFO ABSTRACT Keywords: Fasciolosis among domestic ruminants has resulted in a decrease in the production of milk products and has Fasciola gigantica occasionally led to the deaths of young ruminants due to of acute infections. This study aimed to discriminate Cytochrome B between the eggs of Fasciola gigantica and other trematode eggs in samples collected from ruminant feces spe- fi Species-speci c primer cimens using PCR-based methods with the new candidate gene Cytochrome B (CYTB). A species-specific primer Epidemiological mapping was developed with a high degree of sensitivity (3.285 pg). The primer was able to amplify the F. gigantica genomic DNA and there were no positive results with the other related trematodes (Paramphistomum sp., Orthocoelium sp., Fischoederius sp., Calicophoron sp., Echinostoma revolutum, E. cinetorchis, E. ilocanum and Isthmiophora hortensis), freshwater snails (Lymnaea auricularia, Bithynia siamensis, Indoplanorbis exustus, Melanoides tuberculata, Tarebia granifera)ordefinitive hosts (Bos primigenius and Bubalus bubalis). The minimum concentration of DNA from eggs that could be give a positive result was 3.285 pg. Moreover, the results of the study confirmed the existence of F. gigantica in Nakhon Pathom Province with a high prevalence (28.57%) and revealed the area of infection through epidemiological mapping. Thus, the species-specific primer and epide- miological data in this study may be helpful for use in epidemiological studies, phylogenetic studies and ve- terinary studies in the future. 1. Introduction including diarrhea, weight loss, decreased milk production and occa- sionally deaths in young ruminants due to acute and heavy infections Domestic and free-living ruminants are frequently infected by var- (Elkhatam and Khalafalla, 2016; Umur et al., 2018). The F. gigantica is ious intestinal, stomach or liver trematodes including digenetic species the dominant species of Fasciola genus in Southeast Asia (Wannasan of the Paramphistomum, Orthocoelium, Fischoederius, Calicophoron and et al., 2014). In order to diagnose F. gigantica infections in both humans Fasciola genera (Dorchies, 2006; Eduardo, 1980; Sanabria and Romero, and ruminants, fecal examination is the classical method that is most 2008; Swarnakar et al., 2014; Zhao et al., 2017). Most of these trema- commonly used to identify eggs in the fecal specimens. However, it is tode species have been induced similar clinical signs (Bazsalovicsová difficult to distinguish between the eggs of F. gigantica and those of et al., 2010). The trematode belonging to the genus Fasciola is a par- other trematodes, such as Paramphistomum (Bazsalovicsová et al., 2010) ticularly important zoonosis trematode that is infectious and causes and Echinostoma (Bless et al., 2015). These eggs display a mophological fasciolosis in both ruminants and humans (Bargues et al., 2016). This apperance that is similar to F. gigantica egg (referred to as F. gigantica- disease effects almost half of the ruminants in the world, has caused like eggs in this study), as these eggs possess very similar sizes, shapes losses amounting to billions of US dollars in the livestock industry, and and internal structures (Bless et al., 2015). has resulted in the deaths of animals in the tropical regions of Africa The validation of the current epidemic area of F. gigantica is im- and Asia (Demerdash et al., 2011; Ichikawa-Seki et al., 2017a; Rast portant for the surveillance or prevention program for the the livestock. et al., 2017). Fasciolosis commonly occurs in the gallbladder and the Several studies have focused on classifying the F. gigantica species by biliary canals of various livestock species such as cattle (Bos primi- molecular data and phylogenetic analysis based on various genes or genius), water buffalos (Bubalus bubalis), goats (Capra aegagrus) and regions, including Cytochrome c oxidase subunit I (CO1) (Ichikawa-Seki other herbivorous ruminants (Ashrafi et al., 2014). The fasciolosis of et al., 2017b), Nicotinamide Adenine Dinucleotide Hydrogen (NADH) de- domestic ruminants are often accompanied by various clinical signs hydrogenase subunit I (ND1) (Hayashi et al., 2016; Ichikawa-Seki et al., ⁎ Corresponding author at: Applied Parasitology Research Laboratory, Department of Biology, Faculty of Science, Srinakharinwirot University, Bangkok, Thailand. E-mail address: [email protected] (T. Chontananarth). https://doi.org/10.1016/j.vetpar.2019.108922 Received 1 March 2019; Received in revised form 26 August 2019; Accepted 7 September 2019 0304-4017/ © 2019 Elsevier B.V. All rights reserved. T. Chontananarth and J. Parawat Veterinary Parasitology 274 (2019) 108922 2016), 28S ribosomal DNA (Raina et al., 2015) and the internal-tran- taxonomic keys (Yamaguti, 1958). scribed spacers 2 (Ai et al., 2010; Chaudhry et al., 2016; Raina et al., 2015). However, to identify the eggs in fecal samples, it has various 2.3. Fecal examination inhibitors that may affect PCR reactions and led to decrease the de- tection limit of PCR reaction (Monteiro et al., 1997). The most suitable The F. gigantica-like eggs in fecal specimens were collected using the genes were selected as a target for a specifically designed and species- modified formalin-ether technique (Allen and Ridley, 1970). This specific primer that was determined to be acceptable for species dis- technique is a concentration method that was designed to separate the crimination among degraded samples. Therefore, a new candidate gene eggs from fecal debris by centrifugation (Allen and Ridley, 1970) and for the identification of F. gigantica eggs was selected. The Cytochrome B has typically been used for trematode egg detection in feces gene (CYTB) holds various informative sites in the nucleotide sequences (Kaewpitoon et al., 2015). First, two grams of the fecal specimens were that are conserved at the genus or species level (Blasco-Costa et al., ground into 10 mL of normal saline solution (NSS). Then, the samples 2016; Valadas et al., 2016). Various reports have used the conserved were placed in the collection tube (15 mL tube; Thermo Fisher Scien- sequences in this gene for the purposes of taxonomic identification of tific, USA) of the centrifuge and spun at a speed of 2500×g for 1 min. several animal species (Ali et al., 2015; Stevanovic et al., 2016; Xu The supernatant was then removed, and all sediment was retained. et al., 2015). Moreover, this gene is suitable for the identification of Subsequently, 10 mL of a 10% formalin was added to the tube. The difficult or degraded specimens, such as those found in processed foods specimens were vigorously shaken and left in the tube for at least 5 min (Ali et al., 2014, 2015) and with degraded DNA, such as that which at room temperature. Next, 3 mL of diethyl ether was added, and the occurs in forensic samples (Staats et al., 2016). Additionally, it has been samples were vigorously shaken for one minute. Finally, the specimens recommended for use as a marker gene because has species specific were put in the collection tube of the centrifuge and spun at a speed of regions that can be separated among trematode species (Blasco-Costa 2000×g for 3 min to separate the solution into 4 layers based on their et al., 2016). Thus, this gene has features that could potentially be used specific gravity (Allen and Ridley, 1970). The layers comprised of ether to construct species-specific primers to amplify the DNA that is present at the top, debris, formalin, and a sediment mixed with parasite eggs at in egg specimens. the bottom. The sediments were used to examine the presence of the Therefore, this study aimed to develop new high-performance spe- eggs under a microscope. All egg specimens were then kept at a tem- cies-specific primers for F. gigantica based on the CYTB gene in order to perature of -20 °C. discriminate between this trematode egg and other F. gigantica-like eggs in the fecal specimens with high specificity. Moreover, this study aimed 2.4. Epidemiological map to investigate the epidemiological situation of F. gigantica in Nakhon Pathom Province by identifying F. gigantica infected areas in Thailand All of F. gigantica-like eggs were separated from other parasitic eggs by using both classical and molecular methods based on PCR techni- based on the morphological characteristics that were observed through ques. This study will then present any differences that are found in the a high magnification compound light microscope (100x). Each of the infection area using both methods. The epidemiological data and the sites infected with F. gigantica-like eggs in Nakhon Pathom Province specific primers obtained from this study could be applied to medical were recorded in order to construct an epidemiological map using the and veterinary studies in order to develop a prevention program of Geographic Information Systems Software (GIS). The infected area was fasciolosis in future studies. indicated by positive results of F. gigantica-like